Ultrapure tetrahydrocannabinol-11-oic acids

ABSTRACT

This application is in the field of medicinal chemistry and relates to ultrapure ajulemic acid, its synthesis, pharmaceutical compositions and methods of use thereof for the treatment and/or prevention of inflammation, pain, and fibrotic diseases including scleroderma, systemic sclerosis, scleroderma-like disorders, sine scleroderma, liver cirrhosis, interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis, Dupuytren&#39;s contracture, keloids, chronic kidney disease, chronic graft rejection, and other scarring-wound healing abnormalities, post-operative adhesions, and reactive fibrosis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Non-Provisional applicationSer. No. 15/220,127 (filed on Jul. 26, 2016), Ser. No. 15/347,059 (filedon Nov. 9, 2016), Ser. No. 15/347,104 (filed on Nov. 9, 2016), Ser. No.15/698,544 (filed on Sep. 7, 2017), Ser. No. 16/198,381 (filed on Nov.21, 2018), and Ser. No. 16/425,186 (filed on May 29, 2019) thedisclosure of each of which is incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

This invention is in the field of medicinal chemistry and relates toultrapure tetrahydrocannabinol-11-oic acids compounds, pharmaceuticalcompositions and synthesis thereof. The invention also relates tomethods of using the compounds and pharmaceutical compositions of thisinvention to treat and/or prevent various conditions such asinflammation, pain and fibrosis.

BACKGROUND OF THE INVENTION

Tetrahydrocannabinol (THC) is the major psychoactive constituent ofmarijuana. In addition to mood-altering effects, THC has been reportedto exhibit other activities, some of which may have therapeutic value,including analgesic, anti-inflammatory and anti-emetic properties. Thepotential therapeutic value of THC has led to a search for relatedcompounds which minimize the psychoactive effects, while retaining theactivities of potential medicinal value.

For example,(6aR,10aR)-3-(1′,1′-dimethylheptyl)-Δ8-tetrahydro-cannabinol-9-carboxylicacid (IUPAC name), also known as ajulemic acid (AJA) is a candidate forthe treatment of pain and inflammation either alone or in combinationwith other agents.

The current body of knowledge of cannabinoid research in pain andinflammation suggests that cannabinoid receptors CB1 and CB2 play animportant role in the initiation and maintenance of post-synapticsignaling and immune mechanisms related to nociception, sensitization,pain signal transmission and pain processing. Previously, impurepreparations of ajulemic acid have been shown to have affinity for bothCB1 and CB2 receptors, with more affinity for the CB1 receptor (14). Thepresent invention, for the first time, provides a highly purified formof ajulemic acid which exhibits a higher affinity for the CB2 receptorthan the CB1 receptor.

The ultrapure ajulemic acid can be used to treat fibrotic diseases suchas scleroderma, systemic sclerosis, scleroderma-like disorders, sinescleroderma, liver cirrhosis, interstitial pulmonary fibrosis,idiopathic pulmonary fibrosis, Dupuytren's contracture, keloids, chronickidney disease, chronic graft rejection, fibrosis of organs such asliver, esophagus, heart, lung, intestines, etc., and otherscarring/wound healing abnormalities, post-operative adhesions, andreactive fibrosis as well as, inflammatory diseases such as lupus,multiple sclerosis, rheumatoid arthritis, dermatomyositis, Marfan'ssyndrome, psoriasis, Type 1 diabetes, diabetes, cancer, asthma, atopicdermatitis, autoimmune thyroid disorders, ulcerative colitis, Crohn'sdisease, Parkinson's disease, Alzheimer's disease, HIV infection, strokeand ischemia, where activation of the CB2 receptor plays a role in thepathophysiology of the disease.

SUMMARY

The present invention provides for a composition comprising ultrapureajulemic acid, wherein the ajulemic acid has an affinity for the CB2receptor greater than its affinity for the CB1 receptor. In someembodiments, ultrapure ajulemic acid has an affinity for the CB2receptor ranging from about 2 times to about 100 times, from about 5times to 50 times, from about 15 times to 50 times, from about 10 timesto about 40 times, or from about 20 times to about 40 times greater thanits affinity for the CB1 receptor. The present invention provides for acomposition comprising ajulemic acid, wherein the ajulemic acid has aK_(i) for the CB1 receptor greater than its K_(i) for the CB2 receptor.In some embodiments, ajulemic acid has a K_(i) for the CB1 receptorranging from about 2 times to about 100 times, from about 5 times to 50times, from about 15 times to 50 times, from about 10 times to about 40times, or from about 20 times to about 40 times greater than its K_(i)for the CB2 receptor. The ajulemic acid in the present composition mayhave purity greater than about 97%, greater than about 98%, or greaterthan about 99%.

The present invention also provides for a composition comprisingajulemic acid, wherein the ajulemic acid has a purity greater than about97%, greater than about 98%, or greater than about 99%.

The present invention provides for a composition comprising ajulemicacid, wherein the ajulemic acid has less than about 1% (w/w), less thanabout 0.5% (w/w), less than about 0.3% (w/w), less than about 0.2%(w/w), less than about 0.1% (w/w), or less than about 0.05% (w/w) of11-hydroxy-(6aR,10aR)-3-(1′,1′-dimethylheptyl)-Δ8-tetrahydrocannabinol(HU-210) or other highly CB1 active compounds.

The ajulemic acid in the present composition may have an affinity forthe CB2 receptor greater than its affinity for the CB1 receptor. In someembodiments, ajulemic acid has an affinity for the CB2 receptor rangingfrom about five times to fifty times, from about ten times to fiftytimes, from about twenty times to about forty times greater than itsaffinity for the CB1 receptor.

The present invention further provides for a method of treating asubject with fibrotic disease comprising administering a therapeuticallyeffective amount of ajulemic acid to the subject, wherein the ajulemicacid has an affinity for the CB2 receptor greater than its affinity forthe CB1 receptor. In some embodiments, ajulemic acid has an affinity forthe CB2 receptor ranging from about 2 times to about 100 times, fromabout 5 times to 50 times, from about 15 times to 50 times, from about10 times to about 40 times, or from about 20 times to about 40 timesgreater than its affinity for the CB1 receptor. The fibrotic disease maybe dermal fibrosis, lung fibrosis, liver fibrosis, kidney fibrosis,heart fibrosis or any other organ fibrosis. The fibrotic disease may bescleroderma, systemic sclerosis, scleroderma-like disorders, sinescleroderma, liver cirrhosis, interstitial pulmonary fibrosis,idiopathic pulmonary fibrosis, Dupuytren's contracture, keloids, cysticfibrosis, chronic kidney disease, chronic graft rejection, or otherscarring/wound healing abnormalities, post-operative adhesions, andreactive fibrosis.

The ajulemic acid may be administered orally, intravenously, topically,ophthalmically, interstitially, by inhalation or via an implant orpatch.

The present invention provides for a method of reducing pain in asubject comprising, administering a therapeutically effective amount ofultrapure ajulemic acid. The ajulemic acid may have an affinity for theCB2 receptor greater than the affinity for the CB1 receptor. In someembodiments, ajulemic acid has an affinity for the CB2 receptor rangingfrom about 2 times to about 100 times, from about 5 times to 50 times,from about 15 times to 50 times, from about 10 times to about 40 times,or from about 20 times to about 40 times greater than its affinity forthe CB1 receptor. The reduction of pain may be measured along at leastone pain scale. For example, the pain may be reduced by at least about 1point, at least about 2 points, at least about 3 points, at least about4 points, at least about 5 points, at least about 6 points, at leastabout 7 points, or at least about 8 points on an 11-point pain scale.

The present invention also provides for a method of reducinginflammation in a subject comprising, administering a therapeuticallyeffective amount of ultrapure ajulemic acid. The ajulemic acid may havean affinity for the CB2 receptor greater than the affinity for the CB1receptor. In some embodiments, ajulemic acid has an affinity for the CB2receptor ranging from about 2 times to about 100 times, from about 5times to 50 times, from about 15 times to 50 times, from about 10 timesto about 40 times, or from about 20 times to about 40 times greater thanits affinity for the CB1 receptor. The reduction of inflammation may bemeasured by at least one inflammation assay.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the structure of ajulemic acid((6aR,10aR)-3-(1′,1′-dimethylheptyl)-Δ8-tetrahydrocannabinol-9-carboxylicacid) and the naturally occurring pentyl side chain analog,(6aR,10aR)-Δ8-tetrahydrocannabinol-9-carboxylic acid.

FIG. 2 shows some critical steps for the synthesis of ultrapure ajulemicacid synthesis.

FIG. 3 shows a scheme for synthesis of ultrapure Ajulemic Acid (AJA).

FIG. 4 shows a comparison of the properties of batches of AJA asdescribed in U.S. Pat. No. 5,338,753 and ultrapure AJA made with5-(1′,1′-dimethylheptyl)-resorcinol (DMHR)-lot JBA1001A04.

FIGS. 5A and 5B show an LC-MS analysis of AJA made with ultrapure5-(1′,1′-dimethylheptyl)-resorcinol (DMHR).

FIG. 6 shows an HPLC analysis of synthesized ultrapure AJA.

FIG. 7 shows a magnified image of an HPLC analysis of synthesizedultrapure AJA.

FIG. 8 shows an analysis of ultrapure AJA showing 99.8% purity.

FIG. 9 shows affinity constants for selected cannabinoids. The ultrapureAJA shows a major difference between the K_(i) for CB1 and CB2receptors.

FIG. 10 shows the cataleptic and antinociceptive effects of AJA fromBurstein, S. H. et al. (1992) Synthetic nonpsychotropic cannabinoidswith potent antiinflammatory, analgesic, and leukocyte antiadhesionactivities, J Med Chem 35(17), 3135-3141.

FIG. 11 shows plasma and brain levels of cannabinoids following systemicadministration in rats as described in Dyson et al. [3].

FIG. 12 shows representative binding curves of selected cannabinoids forCB2 and CB1.

FIG. 13 demonstrates effects of CP55940 (circles) and JBT-101 (ultrapureAJA; squares) on [₃₅S]GTPγS turnover in hCB₁ and hCB₂ receptors (upperand lower panels, respectively) expressed in HEK-293 cells. Eachconcentration-effect curve represents the mean (±SEM) of 4 repetitions.Conditions used were adapted from Wiley et al. (20).

FIG. 14 shows that JBT-101 is active in the ring test only at high dosesof 30 mg/kg and above. All drugs were given orally in oil. Conditions asreported by Wiley J L and Martin B R (21). This effect is considered tobe CB1 mediated.

FIG. 15 shows that JBT-101 is active in the mouse hot plate assay onlyat high doses of 30 mg/kg and above. By comparison, HU-239 (i.e., AJAreported in U.S. Pat. No. 5,338,753) was active at low doses givenorally (<0.5 mg/kg). MPE: maximum possible effect. Experimentalconditions as reported in Burstein, et al. (22).

FIG. 16 shows that JBT-101 is inactive in the mouse hypothermia assay.All drugs were given orally in oil. Experimental conditions as reportedby Wiley J L and Martin B R (21). This effect is considered to beCB1-mediated.

FIG. 17 shows the CB2 specificity of ultrapure JBT-101 in stimulation ofPGJ in HL-60 cells. The CB2 antagonist SR144528 reduced ultrapureAJA-induced PGJ synthesis in HL60 immune system cells at lowconcentrations (squares). The CB1 antagonist SR141716 has a much smallereffect (triangles). DMSO control (open circles). Treatments of cells wascarried out in 48 well plates with 20,000 cells/500 μl RPMI/FCSmedia/well. Cells were incubated for 20 hrs at 37° C. and 5% CO₂. Mediawas changed to 500 μl of serum free RPMI and TNFα (10 nM) added. Treatedfor 2 hrs with SR144528 [1 μM] or SR141716 [10 μM] and 100 μl removedfor ELISA assays. N=4.

FIG. 18 shows that JBT-101 is effective at inhibiting skin fibrosis inthe bleomycin mouse model of scleroderma, a CB2 dependent response. Micewere treated daily with local injection of bleomycin and administeredorally the indicated doses of JBT-101 (ultrapure AJA). All doses,including a dose of 1 mg/kg (data not shown) were equally effective atinhibiting skin thickening.

FIG. 19 shows that JBT-101 (ultrapure AJA) is effective at doses wellbelow the doses in which CB1 activity is observed, at inhibiting pawvolume in a model of arachidonic acid induced paw edema, a CB2 dependentresponse. Mice were administered orally the indicated doses of JBT-101(ultrapure AJA) followed 90 minutes later with intraplanar injection ofarachidonic acid in the right paw. Right paw volume was measured 45minutes post-arachidonic acid injection.

DETAILED DESCRIPTION OF THE INVENTION THC Derivatives

Tetrahydrocannabinol (THC) is the major psychoactive constituent ofmarijuana. In addition to mood-altering effects, THC has been reportedto exhibit other activities, some of which may have therapeutic value,including analgesic, anti-inflammatory and anti-emetic properties. Thepotential therapeutic value of THC has led to a search for relatedcompounds which minimize the psychoactive effects, while retaining theactivities of potential medicinal value.

For example,(6aR,10aR)-3-(1′,1′-dimethylheptyl)-Δ8-tetrahydro-cannabinol-9-carboxylicacid, also known as ajulemic acid, may be used for the treatment of painand inflammation either alone or in combination with other agents.

The current body of knowledge of cannabinoid research in pain andinflammation suggests that CB1 and CB2 receptors play an important rolein the initiation and maintenance of post-synaptic signaling and immunemechanisms related to nociception, sensitization, pain signaltransmission and pain processing. [C. Voscopoulos and M. Lema, Br. J.Anaesth. (2010) 105 (suppl 1): i69-i85.]. Previously, earlierpreparations of ajulemic acid have been shown to have affinity for bothCB1 and CB2 receptors, with higher affinity for the CB1 receptor. Thepresent invention, for the first time, provides for a purified form ofajulemic acid with higher affinity for the CB2 receptor vs the CB1receptor. The purified form of ajulemic acid is also referred to asultrapure ajulemic acid.

In various embodiments, the degree of purity of the ajulemic acid isgreater than about 95% (w/w), greater than about 96% (w/w), greater thanabout 97% (w/w), greater than about 98% (w/w), greater than about 99% v,greater than about 99.1% (w/w), greater than about 99.2% (w/w), greaterthan about 99.3% (w/w), greater than about 99.4% (w/w), greater thanabout 99.5% (w/w) or greater than about 99.9% (w/w). The degree ofpurity may be assessed by a variety of different methods as describedfurther below.

The affinity of the purified form of ajulemic acid for the CB2 receptorcan range from about five-fold to about ten-fold greater than theaffinity for the CB1 receptor, but affinity ranges from about 5×-50×,7×-10×, 8×-15×, 10×-20×, 15×-30×, 25×-50×, 40-75× and 50×-100× are alsoencompassed by the invention (ranges represent ratios of affinity ofajulemic acid for the CB2 receptor versus the CB1 receptor).

In one embodiment, the present compound has the structure shown inFormula I. The present compounds may have a purity of greater than about97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about99.4%, about 99.5% or about 99.9%. The present compounds may containless than 0.1% (w/w) of11-hydroxy-(6aR,10aR)-3-(1′,1′-dimethylheptyl)-Δ8-tetrahydrocannabinol(HU-210) or other highly CB1 active compounds. The purified form of thepresent compounds may also be referred to as an ultrapure form. Alsoencompassed by the present invention are the pharmaceutically acceptablesalts, esters, or solvate of the compound in Formula I.

where R₁ is hydrogen, COCH₃ or COCH₂CH₃; R₂ is a branched C5-C12 alkylgroup which may optionally have a terminal aromatic ring, or optionallya branched OCHCH₃(CH₂)_(m) alkyl group which may have a terminalaromatic ring and where m is 0 to 7; R₃ is hydrogen, a C1-8 alkyl or aC1-8 alkanol group; and Y is either zero, i.e. absent, or a bridginggroup of NH or oxygen, (provided that where Y is oxygen and R₂ is abranched C5-C12 alkyl, R₃ is not CHCH₃).

Preparation of Ultrapure Ajulemic Acid

The present invention provides for a process of preparing a purifiedform of ajulemic acid. The process may contain the following steps: (a)reacting Para-mentha-2,8-dien1-ol (PMD) and 5-(1,1-dimethylheptyl)resorcinol (DMHR) to form(6aR,10aR)-3-(1′,1′-dimethylheptyl)-Δ8-tetrahydrocannabinol (Compound8); (b) acetylating Compound 8 to form(6aR,10aR)-3-(1′,1′-dimethylheptyl)-Δ8-tetrahydrocannabinol acetate(Compound 9); (c) oxidizing Compound 9 to form11-oxo-(6aR,10aR)-3-(1′,1′-dimethylheptyl)-Δ8-tetrahydrocannabinolacetate (Compound 10); (d) oxidizing Compound 10 to form(6aR,10aR)-3-(1′,1′-dimethylheptyl)-Δ8-tetrahydrocannabinol-9-carboxylicacid acetate (Compound 11) using hydrogen peroxide, wherein the molarratio of hydrogen peroxide to Compound 9 ranges from about 2:1 to about7:1; (e) hydrolyzing Compound 11 to produce crude ajulemic acid; (f)acetylating crude ajulemic acid to form Compound 11; and, (g)hydrolyzing Compound 11 to form the purified form of ajulemic acid.

In step (d), the molar ratio of hydrogen peroxide to Compound 9 may alsorange from about 2:1 to about 6:1, from about 2:1 to about 5:1, fromabout 2:1 to about 4:1, about 2:1, about 2.5:1, about 3:1, about 3.5:1,or about 4:1. In step (a), the molar ratio of PMD to DMHR may range fromabout 1:1 to about 3:1, from about 1:1 to about 2:1, from about 1:1 toabout 1.1:1, about 1.1:1, or about 1.2:1. Step (a) may be carried out atabout 50° C. to about 120° C., about 60° C. to about 110° C., about 70°C. to about 100° C., about 75° C. to about 90° C., about 70° C. to about80° C., about 70° C., about 75° C., or about 80° C. The purifiedajulemic acid may have a purity greater than about 95% (w/w), greaterthan about 96% (w/w), greater than about 97% (w/w), greater than about98% (w/w), greater than about 99% v, greater than about 99.1% (w/w),greater than about 99.2% (w/w), greater than about 99.3% (w/w), greaterthan about 99.4% (w/w), greater than about 99.5% (w/w) or greater thanabout 99.9% (w/w).

In certain embodiments, the compounds of the invention contain one ormore chiral centers. The term “purity” can also encompass chiral purity.The purity of a stereoisomer of ajulemic acid refers to chemical purityand/or chiral purity of the stereoisomer. For example, the purity ofajulemic acid can include both the chemical purity and the chiral purityof ajulemic acid. The chiral purity of a stereoisomer of ajulemic acidmay be greater than about 98.5% (w/w), greater than about greater thanabout 95% (w/w), greater than about 96% (w/w), greater than about 97%(w/w), greater than about 98% (w/w), greater than about 99% v, greaterthan about 99.1% (w/w), greater than about 99.2% (w/w), greater thanabout 99.3% (w/w), greater than about 99.4% (w/w), greater than about99.5% (w/w) or greater than about 99.9% (w/w).

The purity of the present compounds may be assayed by gas chromatography(GC) or high pressure liquid chromatography (HPLC). Other techniques forassaying the purity of ajulemic acid and for determining the presence ofimpurities include, but are not limited to, nuclear magnetic resonance(NMR) spectroscopy, mass spectrometry (MS), GC-MS, infrared spectroscopy(IR), thin layer chromatography (TLC), and differential scanningcalorimetry. Chiral purity can be assessed by chiral GC or measurementof optical rotation.

The purified form of ajulemic acid may be stable after storage. Forexample, after storage at about 5° C. for at least 3 months, the presentcomposition may contain greater than about 98.5% (w/w), greater thanabout 99% (w/w), greater than about 99.5% (w/w), or greater than about99.9% (w/w) ajulemic acid. After storage at 25° C. and 60% relativehumidity for at least 3 months, the present composition can containgreater than about 98.5% (w/w), greater than about 99% (w/w), greaterthan about 99.5% (w/w), or greater than about 99.9% (w/w) ajulemic acid.

Several embodiments of the process are described below. They arepresented for the purposes of illustration only and are not limiting theinvention.

Purification A. Optimization of Allylic Oxidation Applied to Synthesisof Ajulemic Acid

The allylic oxidation of Compound 9 containing a methyl at the 11position with selenium dioxide followed by hydrogen peroxide provides away of synthesizing Ajulemic acid to completion without yieldingincompletely oxidized intermediates such as the alcohol at the 11position which gives the highly CB1 active HU-210. Initial laboratoryexperiments using 8 equivalents of hydrogen peroxide, relative toCompound 9, indicated that an adequate conversion was achieved in 4-6hr⁴, see FIG. 2.

Because oxidation reactions are potentially hazardous especially whenconducted at a large scale, safety assessments are performed. The numberof equivalents of hydrogen peroxide was varied from 2, 2.5, 3 and 4equivalents⁵. The thermal onset temperature with these reducedequivalents of hydrogen peroxide did not change from the onsettemperature of 55° C. observed with 8 equivalents, but the maximum selfheat rate when the reduced equivalents of hydrogen peroxide were usedwas just 7° C./min which is significantly reduced from the previouslyobserved 1000° C./min measured when 8 equivalents of hydrogen peroxidewere used. In the case when 2 equivalents of hydrogen peroxide wereused, no thermal event was observed, however reaction completion was notachieved, with a 74.1% conversion of the aldehyde to acid being achievedafter 45 h which did not meet the specification of ≤10.0% of thealdehyde. For the 2.5, 3 and 4 equivalents, reaction completion wasachieved, however, with these numbers of equivalents of hydrogenperoxide, there was still a potential for a runaway reaction.

Therefore calculations were performed, assuming the use of 4 equivalentsof hydrogen peroxide, to determine the rate of addition of chilled waterand the volume required to quench the reaction in the event of a thermalrunaway. An adequate rate of addition and volume of chilled water wasdetermined for controlling a thermal runaway at a rate of 7° C./min, andprotocols implemented for running a 400 g non-GMP batch using 3equivalents of hydrogen peroxide, with pre-chilled water being availablein the event of a thermal runaway. No uncontrollable thermal event wasobserved during the execution of this step in the production run. Animproved yield was also observed over the 3 steps with an increase from16% to 21% yield.

There are several advantages over prior art. While the reagents used inthe synthesis have not changed, it has been demonstrated that the priorart, using 8 equivalents of hydrogen peroxide¹, was operating too closeto a potential catastrophic event. By conducting safety evaluations andlooking at reduced equivalents of hydrogen peroxide, a safely scalableprocess for the allylic oxidation reaction used in the synthesis ofultrapure Ajulemic Acid has been defined, while also improving the yieldfrom 16% to 21%.

B. Improved Synthesis of Ajulemic Acid from DMHR and PMD

Ultrapure DMHR (5-(1,1-dimethylheptyl)resorcinol)) may be purchased fromNorac Pharma (Azusa, Calif.).

Improved Synthetic Procedure and Safety Step 1 Preparation of(6aR,10aR)-3-(1′,1′-dimethylheptyl)-Δ8-tetrahydrocannabinol

Firstly, the amount of PMD (7a in FIG. 3) used in the reaction wasreduced from 1.25 equivalents to 1.1 equivalents, because the 0.1equivalents excess of PMD was sufficient to react with all of the DMHR(6 in FIG. 3).

The previous procedure describes heating the batch to reflux (˜110° C.)for 3 h. Although effective in azeotropically removing water and meetingthe specification of ≤2.0% (AUC) cannabidiol, this procedure inducedCompound 1 decomposition and DMHR regeneration upon prolonged heating.To avoid byproduct formation, the reaction was conducted at 75° C., thesame temperature as the subsequent acetylation reaction, and was stableover 24 h with no effects on reaction time. In order to azeotropicallyremove water, the reaction was placed under partial vacuum. A Dean-Starktrap was used to collect the water and the endpoint was redefined as thepoint in which no more water was being collected in the trap.

Lastly, the crystallization conditions were re-examined. Solubilitystudies revealed that the most effective isopropyl alcohol (IPA):waterratio to improve yield was in the range of 3:1 to 5:1 IPA:water. Thiscontrasted the previous procedure, which employed a 5.33:1 ratio. Afterexperimentation with different ratios, 8:2 IPA:water was used as thesolvent ratio for crystallization. Also, crystal size was improved byslowing the stirring rate, which kept the larger crystals intact.

Seed crystals were no longer required for crystallization because theAcetylated PMD/DMHR coupled product consistently crystallizes followingthe procedure described.

Step 2 Preparation of(6aR,10aR)-3-(1′,1′-dimethylheptyl)-Δ8-tetrahydrocannabinol-9-carboxylicacid acetate

The previous procedure used 3 equivalents of hydrogen peroxide for theoxidation reaction. While this was much safer than the 8 equivalentsused in another previous procedure, there was still a possibility of athermal runaway. Accordingly, 2 equivalents of hydrogen peroxide wasevaluated, because previous safety studies showed that 2 equivalents ofhydrogen peroxide drastically reduced the risk of thermal runaway. After48 h, the reaction did not meet the specification of <10% (AUC) ofunreacted aldehyde. Regardless, the reaction was taken through to CrudeAjulemic acid (6.2% yield, 96.8% (AUC) purity). Even though the puritywas comparable, the yield was significantly lower than the reaction with3 equivalents of hydrogen peroxide. 3 equivalents of hydrogen peroxidewas used and thus close technical supervision of this step duringproduction can ensure good temperature control. Also, as previously donefor the 400 g non-GMP batch, chilled water was present as a quenchoption in the event of an uncontrolled exothermic reaction.

Toluene was previously used as an extraction solvent after the hydrogenperoxide oxidation. The procedure then required evaporating the mixtureto dryness and adding heptane. Since evaporation to dryness is noteasily scalable and a solvent swap from toluene to heptane is notefficiently feasible, other solvents were evaluated as possibleextraction solvents. Firstly, heptane was assessed. Although heptanesuccessfully extracted the product into the organic layer, the phasesplit was slow and there were three layers. On the second attempt, noextraction solvent was used and it was observed that quenching thehydrogen peroxide reaction with 20 wt % sodium thiosulfate led to twophases. Thus, the aqueous layer could be removed without additionalorganic solvents.

During the synthetic process development, the phase split following thehydrolysis was problematic because of the formation of 3 layers, thehigh viscosity of the middle layer, and the dark color of all thephases. MTBE was evaluated as a possible extraction solvent instead ofheptane. Although the MTBE extraction, led to a quick phase split into 2layers—a dark brown organic layer and a transparent red aqueous layer,HPLC analysis revealed that the product and all of the impuritiesremained in the organic layer. Heptane was then re-evaluated as theextraction solvent. As observed in the 400 g batch production, thereaction mixture separated into 3 dark layers; however the middle oilylayer was much more mobile (believed to be due to THF being present).HPLC analysis showed that the top organic layer contained impurities andno product while the two bottom layers contained mostly product withtrace impurities. Although the phase split was still hard to visualizedue to the dark color, the heptane extraction was able to separate theproduct from the impurities.

The subsequent acidification and extraction steps were performed atambient temperatures instead of 45-55° C. These conditions reduced therisk of hydrolyzing MTBE to produce chloromethane and tert-butanol.

The next step was to filter the batch through Celite to break upemulsions. Because the filtration was noticeably slow, the aqueous layerwas removed prior to filtration. The solvent swap from MTBE toacetonitrile was successful and seed crystals were no longer requiredfor crystallization because the Crude Ajulemic acid consistentlycrystallizes following the procedure described.

Step 3 Preparation of Crude Ajulemic Acid

In order to avoid a difficult solvent swap from toluene to heptane, theacetylation reaction was completed in heptane at 45-55° C. The changewas successful on a small scale, but the reaction mixture solidifiedafter ˜0.5 equivalents of pyridine was charged to the reactor at a 14.5g scale. To try to recover the Ajulemic acid, MTBE was added tosolubilize the precipitate and HCl was used to remove the pyridine. Thisattempt was unsuccessful, because the solids precipitated out whileevaporating the solvent on the rotary evaporator. NMR analysis revealedthat the precipitate was an Ajulemic acid-pyridine partial salt. Severalexperiments were completed in order to determine the cause of the saltformation. Firstly, the same reaction was performed on a smaller scale(˜2.5 g). Pyridine (0.5 equivalents) was slowly added to the mixture.Once again, the reaction solidified. This, however, was remedied uponadding the full 2.1 equivalents of pyridine. The reaction was thenperformed at 25° C. and 75° C., again adding 0.5 equivalents beforeadding the full amount of pyridine. In both cases, the salt precipitatedbut solubilized once all of the pyridine was added. Since a larger batchsize will require slower additions, this procedure was deemedun-scalable.

Accordingly, crude Ajulemic acid dissolved in heptane (6 equivalents)was added to a reactor containing the full 2.1 equivalents of pyridinedissolved in heptane (2 equivalents). This avoided the formation of apartial salt because pyridine was always in excess. This also increasedthe reaction volume to 10 equivalents heptane (2 extra equivalents wasused to wash the reactor), which improved mobility duringcrystallization.

Step 4 Preparation of Ultrapure Ajulemic Acid

Similar changes were made in this step as in the Step 2 work-up as thesame reaction was being performed. The acidification of the reactionmixture was done at ambient temperature to avoid MTBE hydrolysis and anextra water wash was added to avoid oven corrosion.

The main issue with Step 4 was the drying time. It required 9 days toreach the specification of ≤410 ppm acetonitrile for the previous 400 gnon-GMP campaign batch. Thus, crystallization from IPA/water wasevaluated. A solubility study showed that Ajulemic acid in a 1:1IPA/water mixture had a similar solubility to Ajulemic acid in a 3:1acetonitrile/water mixture. Although Ajulemic acid was successfullyisolated from IPA/water, the drying time did not improve, requiring >7days to reach the specification of ≤5000 ppm IPA on a 2.5 g scale. Theamount of IPA remaining in the product did not correlate with the LOD,suggesting that water played a key role in drying time. Therefore,crystallization from pure acetonitrile was evaluated on a 19.5 g scale.This produced a much shorter drying time with no detectable amount ofacetonitrile at 81 h.

A use-test of ultrapure DMHR (purity of 99.4% (AUC)) was successfullytaken through to Ajulemic Acid. The purity of the Ajulemic acid was99.9% (AUC) with no single peak ≥0.04%.

C. The Synthesis of Ajulemic Acid from Ultrapure DMHR and PMD (LargeScale)

Step 1

To a 200-gallon reactor were charged ultrapure DMHR (20.0 kg, 1 equiv.),PTSA (3.40 kg, 0.2 equiv.) and toluene (102.3 kg, 5 vol. equiv.). Tothis was added PMD (14.18 kg, 1.1 equiv.) over 38 min, followed by atoluene rinse (17.4 kg, 1 vol. equiv.) while maintaining the batchtemperature at 15-30° C. The batch was heated to 70-80° C. under partialvacuum, and a Dean-Stark trap filled with toluene was used to removewater by azeotropic distillation with toluene while maintaining aconstant volume of toluene. The reaction was determined complete after 2h by HPLC, detecting no cannabidiol and obtaining a Δ8:Δ9 ratio of 106:1(specification was ≤2.0% (AUC) cannabidiol and Δ8:Δ9 ratio≥4:1). Thebatch was held overnight at 25° C. and atmospheric pressure.

The batch was reheated to 70-80° C., and pyridine (10.70 kg, 1.6 equiv.)and acetic anhydride (13.80 kg, 1.6 equiv.) were each added over ˜30 minwhile maintaining the batch temperature at 70-80° C. After 2 h the batchwas sampled and passed the specification of ≤2.0% (AUC) of Compound 1with 0.4% (AUC) Compound 1 detected. Water (160.0 kg, 8 equiv.) wasadded and the batch was adjusted to 50-60° C. The lower aqueous layerwas removed and the batch was further washed with water (40 kg, 2.0equiv.) at 50-60° C. The reaction mixture was transferred from the200-gallon reactor to the 250-liter reactor.

Toluene (100 L, 5 vol. equiv.) was distilled off and IPA (78.6 kg, 8vol. equiv.) was added. This was repeated two more times before thesample was tested and passed the specification of ≤2.0% (AUC) oftoluene. The batch was held at 20-30° C. overnight. After IPA (31.4 kg,2 vol. equiv.) addition, the batch was reheated to 45-55° C., water(40.0 kg, 2 vol. equiv.) was added, and the temperature was maintainedfor an additional ˜1 h before cooling to 25±2° C. at −10° C./hour. Thebatch was held at this temperature for over 16 h then cooled to 0-5° C.at 10° C./hour and held for another 2 h 10 min. The slurry containedlarge particles that would not flow through the bottom outlet valve.Therefore, the product was dissolved back into solution by heating thereactor to 55° C. The batch was cooled to 35° C., held at thistemperature overnight, and filtered to yield a 1st crop. The filtratewas then cycled back into the reactor, cooled down to 5° C. and furtherproduct was isolated as 2nd crop. Both crops were washed with 20% waterin IPA solution (˜36 L used to wash 1st crop and ˜24 L for 2nd crop).The two crops were then dried under vacuum at 122° F. (50±5° C.). Theproduct was offloaded and gave a total actual weight of 25.76 kg ofCompound 2 The 1st crop yielded an actual weight of 14.50 kg (97.0%(AUC) purity) and the 2nd crop yielded an actual weight of 11.26 kg(93.3% (AUC) purity).

Step 2

To a 200-gallon reactor was charged Compound 2 (25.76 kg, 1 equiv.),selenium dioxide (8.66 kg, 1.25 equiv.), tetrahydrofuran (98.5 kg, 4.3vol. equiv.) and water (5.2 kg, 0.2 equiv.). The reactor was heated andmaintained at 55-65° C. After 20.5 h, the reaction was deemed completeby HPLC, passing the specification of ≤2.0% (AUC) Compound 2 with 1.8%(AUC) residual Compound 2. The batch was cooled to 0-10° C. over ˜3 h.While maintaining the batch temperature <25° C., 35 wt % hydrogenperoxide (18.21 kg, 3 equiv.) was added. The batch temperature was thenadjusted to 10-25° C. and held at this temperature until the reactionmet the specifications of ≤10.0% (AUC) of Compound 10 by HPLC. At ˜16 h,the reaction was deemed complete (1.7% (AUC) residual Compound 10) andwas slowly quenched with 20 wt % sodium thiosulfate solution (98.8 kg, 2equiv.) while maintaining the batch temperature <35° C. After 2 h 13min, there was no trace of peroxide. The batch was filtered through apad of Celite and the Celite cake was washed with 22.9 kg THF (1 vol.equiv.). The phases were allowed to separate and the aqueous layer wasdrained. The reaction mixture was then washed with 10 wt % sodiumchloride (51.6 kg, 2 equiv.) and transferred back into the cleaned200-gallon reactor along with water (128.8 kg, 5 equiv.). To this wasadded 50 wt % sodium hydroxide solution (18.8 kg, 3.8 equiv.) whilemaintaining the batch temperature <55° C. The batch was held at 45-55°C. for ˜1 h after which time it was sampled and met the specification of≤2.0% (AUC) of Compound 11 by HPLC, detecting 0.1% (AUC) residualCompound 11.

The reaction was allowed to cool to 25±2° C. and heptane (44.1 kg, 2.5vol. equiv.) was added. The reaction mixture was stirred for 30 min andsettled for 48 min. Three phases were observed: bottom red/brown aqueouslayer, middle viscous black layer, and top clear red organic layer. Thetop organic layer was removed and the middle and bottom productcontaining layers were combined and washed with another 44.1 kg (2.5vol. equiv.) of heptane. Once again, three phases were observed and thetop organic layer was removed.

While maintaining the temperature ≤30° C. the pH of the combined middleand bottom layers was adjusted to pH<1.5 using 37 wt % hydrochloric acid(24.38 kg). MTBE (66.8 kg, 3.5 vol. equiv.) was added, and the mixturewas stirred for 30 min before allowing to settle for 20 min. Upon tryingto drain the lower aqueous phase, it was observed that the phases hadnot fully separated. Through experimentation with a small aliquot of thereaction mixture (taken earlier for pH testing), more MTBE (66.7 kg, 3.5vol. equiv.) was added to the reactor. After stirring for 30 min andallowing the reaction mixture to settle for an additional 2 h, the loweraqueous phase was drained, producing a clean phase separation. Thereaction mixture was washed with water (51.5 kg, 2 equiv.) and heldovernight for 15 h 10 min before draining the lower aqueous layer. Thereaction mixture was filtered through a pad of Celite, and the cake waswashed with MTBE (9.53 kg, 0.5 vol. equiv.). The batch was transferredinto the 250-liter reactor and 160 L (˜6.2 vol. equiv) of solvent wasremoved by distillation. [Note: The distillation volume was adjusted toaccount for the extra MTBE added.] Acetonitrile (30.4 kg, 1.5 vol.equiv.) was added and 38-42 L (˜1.5 vol. equiv.) of solvent was removed.This was repeated three times. The batch was cooled to 0-5° C. at ˜10°C./h, held at temperature for 2 h, and filtered. The cake was thenwashed with 30.4 kg of pre-chilled acetonitrile (1.5 vol. equiv). Afterthe cake was deliquored and dried at 122° F. (50±5° C.), the product wasoffloaded to yield crude Ajulemic Acid (5.29 kg actual weight, 21.1%yield, purity: 99.0% (AUC) by HPLC using Method A: see Table 1) as anoff-white solid.

TABLE 1 ASSAY AND RELATED SUBSTANCES CHROMATOGRAPHIC CONDITIONS The HPLCsystem is operated in the gradient mode. Column Agilent Zorbax SB-CN, 5μm, 4.6 × 250 mm, P/N: 880975-905 Flow 1.5 mL/min Injection Volume 10 μLAutosampler Ambient Temperature Column Temperature Ambient DetectionWavelength 220 nm Run Time 37 min Pump A Water, HPLC grade Pump B 0.1%Phosphoric Acid in Acetonitrile:Methanol (90:10) Needle Wash MethanolGradient Program Time (min) Pump A (%) Pump B (%) 0 55 45 20 45 55 30 0100 30.1 55 45 37 55 45

Step 3

To a 40-liter reactor was charged crude Ajulemic Acid (5.28 kg, 1equiv.) and heptane (21.8 kg, 6 vol. equiv.). To a 250-liter reactor wascharged pyridine (2.18 kg, 2.1 equiv.) and heptane (7.2 kg, 2 vol.equiv.). Both reactors were then heated to 50-60° C. After the contentsof the 40-L reactor had dissolved the solution was transferred to the250-L reactor and additional heptane (7.2 kg, 2 vol. equiv.) was used torinse the 40-L reactor with this being transferred to the 250-L reactor.While maintaining the batch temperature at 50-60° C., acetic anhydride(2.50 kg, 1.8 equiv.) was added and the reaction mixture was stirred for2 h. The reaction mixture was sampled and showed 0.8% (AUC) crudeAjulemic Acid, which did not meet the specification of ≤0.5% (AUC). Asecond sample was obtained after 3 h which passed the specification with0.2% (AUC) crude Ajulemic Acid detected.

To the reactor was then slowly charged deionized (DI) water (7.40 kg,1.4 equiv.) while maintaining the temperature at 50±5° C. The reactionmixture stirred for 2 h and analyzed by HPLC to meet the specificationof ≤0.5% Acetylated Ajulemic Anhydride (AUC) by HPLC with 0.3% (AUC)Acetylated Ajulemic Acid being detected. The lower aqueous layer wasdrained and the organic phase was washed with 1N HCl (14.80 kg, 2.8equiv). The pH of the aqueous layer was 3 and met the specification ofpH≤5. The organic layer was washed once more with water (7.40 kg, 1.4equiv.) to obtain pH 4 which passed the specification of pH≥3. The batchwas allowed to cool to 0-5° C. at −10° C./h overnight, followed byholding for 3 h at 0-5° C. The batch was filtered, washed withpre-cooled heptane (11.2 kg, 3 equiv.), and dried under vacuum at 122°F. (50±5° C.). The product was offloaded and gave Acetylated AjulemicAcid (5.03 kg actual weight, 86.1% yield) as a white solid with a purityof 99.2% (AUC) by HPLC (Method A).

Step 4

To a 40-liter reactor was charged Acetylated Ajulemic acid (5.02 kg, 1equiv.), MTBE (15.37 kg, 4.13 vol. equiv.), and 2N NaOH (14.54 kg, 2.400equiv.) while maintaining the batch temperature ≤50° C. The batch wasmaintained at 45-55° C. for 4 h 2 min after which time the batch had nounreacted Acetylated Ajulemic Acid and met the specification of ≤0.5%(AUC) Acetylated Ajulemic Acid by HPLC. The reaction mixture was allowedto cool to 25° C. While maintaining the batch temperature 25±5° C., thebatch was acidified with 37 wt % HCl (3.62 kg, 0.60 vol. equiv.). Afterstirring for 30 min the lower aqueous layer was separated and theorganic layer was washed with water (5.53 kg, 1.1 equiv.). The pH was 2.Two additional water washes were performed and the reaction mixturereached pH 3. Although this did not meet the specification of pH>3, thereaction was continued.

The organic layer was filtered through Celite, a 10-micron filter, and a2.4 micron filter into a 250-liter reactor. The 40-liter reactor wasthen washed with MTBE (1.86 kg, 0.5 vol. equiv.) and the batch alongwith the wash was transferred back into the cleaned 40-liter reactorthrough a 10-micron and a 2.4 micron filter. MTBE (10 L, 2 vol. equiv.)was distilled off and acetonitrile (17.4 kg, 4.4 vol. equiv.) was added.Then, 22.25 L (˜4.4 vol. equiv.) of solvent was distilled off, and thedistillate was analyzed by NMR to reveal 46% MTBE. Finally, additionalacetonitrile (17.4 kg, 4.4 vol. equiv.) was added and 14.6 L (2.9 vol.equiv.) solvent was distilled off. The distillate was analyzed by NMR toreveal 0.6% MTBE. The temperature was adjusted to 20-30° C. over 9 h 25min. Crystals were present and reaction mixture was cooled to 0-5° C.over 2 h. It was held for 3 h before the contents were filtered. Themother liquor was recirculated through the reactor several times tofacilitate slurry transfer. The crystals were washed with 9.8 kg (2.5vol. equiv.) of pre-chilled acetonitrile. The product dried in thevacuum oven at 122-130° F. with a slight nitrogen bleed. After 258 hr,the product was assayed by GC and met the specification of ≤200 ppm. Theproduct was offloaded and gave Ajulemic Acid (4.12 kg actual weight,90.7% yield) with a purity of 99.8% (AUC) by HPLC (Method A).

Receptor Binding

Affinity, or binding affinity, is a measure of the strength of thebinding interaction between two or more distinct molecular entities(e.g. between a compound and a receptor) that can be defined byequilibrium binding constants or kinetic binding rate parameters.Examples of suitable constants or parameters and their measurement unitsare well known in the art and include, but are not limited to,association constant (K_(A)); dissociation constant (K_(D)) orinhibition constant (K_(i)); association rate constant (K_(on)) anddissociation rate constant (K_(off)). In one embodiment, K_(i) equals[receptor]·[inhibitor]/[receptor bound by inhibitor], so K_(i) is anequilibrium constant for the inhibitor binding to the receptor. In thecase of K_(A), higher values mean stronger or greater binding affinity.In the case of K_(i) (or K_(D)), lower values mean a stronger or greaterbinding affinity.

The present compounds (e.g., the ultrapure AJA) have a greater affinityfor the CB2 receptor than for the CB1 receptor. This means that thepresent compounds (e.g., the ultrapure AJA) bind more tightly to CB2than CB1, i.e., with a smaller K_(i)(CB2) (i.e., K_(i) for the CB2receptor) than its K_(i)(CB1) (i.e., K_(i) for the CB1 receptor).Likewise, the present compounds (e.g., the ultrapure AJA) have a lesseraffinity for the CB1 receptor than for the CB2 receptor. In other words,the present compounds (e.g., the ultrapure AJA) bind less strongly toCB1 than CB2, i.e., with a larger K_(i)(CB1) than its K_(i)(CB2).

The affinity of the purified form of ajulemic acid for the CB2 receptorcan range from about 5 to about 10-fold greater than the affinity forthe CB1 receptor. Ranges from about, 5×-about 50×, about 7×-about 10×,about 8×-about 15×, about 10×-about 20×, about 15×-about 30×, about25×-about 50×, about 40-about 75×, about 50×-about 100×, about 2×-about1000×, about 2×-about 800×, about 5×-about 600×, about 10×-about 500×,about 15×-about 300×, about 5×-about 200×, about 10×-about 100×, about20×-about 80×, or about 10×-about 50× are also encompassed by theinvention (ranges represent ratios of affinity of ajulemic acid for CB2receptor vs. affinity for CB1 receptor, e.g., K_(i)(CB1)/K_(i)(CB2).

In some embodiments, the K_(i)(CB1) (i.e., K_(i) for the CB1 receptor)of the present compounds (e.g., the ultrapure AJA) may be at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 8-fold, at least about 10-fold, at least about50-fold, at least about 100-fold, at least about 200-fold, at leastabout 400-fold, at least about 500-fold, at least about 1000-fold, atleast about 10,000-fold, from about 2-fold to about 10,000-fold, fromabout 2-fold to about 1,000-fold, from about 5-fold to about 500-fold,from about 5-fold to about 100-fold, from about 7-fold to about 10-fold,from about 8-fold to about 15-fold, from about 10-fold to about 20-fold,from about 15-fold to about 30-fold, from about 25-fold to about50-fold, from about 40-fold to about 75-fold, or from about 50-fold toabout 100-fold, of the K_(i)(CB2) (i.e., K_(i) for the CB2 receptor).

The ratio of K_(i)(CB1)/K_(i)(CB2) of the present compounds (e.g., theultrapure AJA) may be at least about 2, at least about 3, at least about4, at least about 5, at least about 8, at least about 10, at least about50, at least about 100, at least about 200, at least about 400, at leastabout 500, at least about 1000, at least about 10,000, from about 2 toabout 10,000, from about 2 to about 1,000, from about 5 to about 500,from about 5 to about 100, from about 7 to about 10, from about 8 toabout 15, from about 10 to about 20, from about 15 to about 30, fromabout 25 to about 50, from about 40 to about 75, or from about 50 toabout 100.

In certain embodiments, the purified form of AJA has a K_(i) for the CB2receptor of about 150 nM or less, about 125 nM or less, about 110 nM orless, about 100 nM or less, about 90 nM or less, about 80 nM or less,about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40nM or less, or about 30 nM or less.

Any conventional method for measuring receptor binding affinity can beused to assay binding of the ligand to the CB1 or CB2 receptor (see,Pertwee R G. Pharmacological action of cannabinoids. Handbook Exp.Pharmacol 168:1-51 (2005); McPartland et al. Meta-analysis ofcannabinoid ligand binding affinity and receptor distribution:interspecies differences. British J. Pharmacology 152:583-593 (2007)).

Binding affinity between two components may be measured directly orindirectly. Indirect measurement of affinity may be performed usingsurrogate properties that are indicative of, and/or proportional to,affinity. Such surrogate properties include: the quantity or level ofbinding of a first component to a second component, or a biophysicalcharacteristic of the first component or the second component that ispredictive of or correlated to the apparent binding affinity of thefirst component for the second component. Specific examples includemeasuring the quantity or level of binding of first component to asecond component at a sub-saturating concentration of either the firstor the second component. Other biophysical characteristics that can bemeasured include, but are not limited to, the net molecular charge,rotational activity, diffusion rate, melting temperature, electrostaticsteering, or conformation of one or both of the first and secondcomponents. Yet other biophysical characteristics that can be measuredinclude determining stability of a binding interaction to the impact ofvarying temperature, pH, or ionic strength.

Binding affinities can be quantified by measuring the rates ofcompound/receptor complex formation and dissociation. Thus, both the “onrate constant” (K_(on)) and the “off rate constant” (K_(off)) can bedetermined by calculation of the concentrations and the actual rates ofassociation and dissociation. (See Nature 361:186-87 (1993)). The ratioof K_(off)/K_(on) is equal to the dissociation constant K_(D). (See,generally, Davies et al. (1990) Annual Rev. Biochem. 59:439-473). K_(i)may be measured by assays such as radioligand binding assays (e.g.,procedures as described in Example 4) or similar assays known to thoseskilled in the art.

The relative affinity for each receptor may be determined by acompetition binding assay between labeled compounds and increasingconcentrations of unlabeled compounds. The binding affinity can bedetermined by competition FACS using labeled compound or othercompetitive binding assays.

The binding affinity of a compound to a receptor can also be determinedby surface plasmon resonance (SPR). K_(D) or K_(i) may be determined bythe BIAcore technology (GE), or the KinExA® (Sapidyne Instruments)affinity analysis.

Conditions to be Treated

The present invention also provides for a method of treating orpreventing conditions described herein by administering to a subject thepresent compound or composition.

Conditions that can be treated or prevented by the present compounds orcompositions include, but are not limited to, fibrotic diseases,inflammatory diseases and pain. Fibrotic diseases include, for example,scleroderma, systemic sclerosis, scleroderma-like disorders, sinescleroderma, liver cirrhosis, interstitial pulmonary fibrosis,idiopathic pulmonary fibrosis, Dupuytren's contracture, keloids, cysticfibrosis, chronic kidney disease, chronic graft rejection, fibrosis oforgans such as liver, esophagus, heart, lung, intestines, etc., andother scarring/wound healing abnormalities, post-operative adhesions,and reactive fibrosis. Inflammatory diseases include, for example,systemic lupus erythematosus, AIDs, multiple sclerosis, rheumatoidarthritis, psoriasis, Type 1 diabetes, diabetes, cancer, asthma, atopicdermatitis, autoimmune thyroid disorders, ulcerative colitis, Crohn'sdisease, neurodegenerative diseases such as Alzheimer's disease,Parkinson's disease, stroke and ischemia.

Non-limiting examples of fibrosis include liver fibrosis, lung fibrosis(e.g., silicosis, asbestosis, idiopathic pulmonary fibrosis), oralfibrosis, endomyocardial fibrosis, retroperitoneal fibrosis, deltoidfibrosis, kidney fibrosis (including diabetic nephropathy), cysticfibrosis, and glomerulosclerosis. Liver fibrosis, for example, occurs asa part of the wound-healing response to chronic liver injury. Fibrosiscan occur as a complication of haemochromatosis, Wilson's disease,alcoholism, schistosomiasis, viral hepatitis, bile duct obstruction,exposure to toxins, and metabolic disorders. Endomyocardial fibrosis isan idiopathic disorder that is characterized by the development ofrestrictive cardiomyopathy. In endomyocardial fibrosis, the underlyingprocess produces patchy fibrosis of the endocardial surface of theheart, leading to reduced compliance and, ultimately, restrictivephysiology as the endomyocardial surface becomes more generallyinvolved. Oral submucous fibrosis is a chronic, debilitating disease ofthe oral cavity characterized by inflammation and progressive fibrosisof the submucosal tissues (lamina propria and deeper connectivetissues). The buccal mucosa is the most commonly involved site, but anypart of the oral cavity can be involved, even the pharynx.Retroperitoneal fibrosis is characterized by the development ofextensive fibrosis throughout the retroperitoneum, typically centeredover the anterior surface of the fourth and fifth lumbar vertebrae.

Scleroderma is a disease of the connective tissue characterized byfibrosis of the skin and internal organs. Scleroderma has a spectrum ofmanifestations and a variety of therapeutic implications. It compriseslocalized scleroderma, systemic sclerosis, scleroderma-like disorders,and sine scleroderma. Systemic sclerosis can be diffuse or limited.Limited systemic sclerosis is also called CREST (calcinosis, Raynaud'sesophageal dysfunction, sclerodactyly, telangiectasia). Systemicsclerosis comprises: scleroderma lung disease, scleroderma renal crisis,cardiac manifestations, muscular weakness including fatigue or limitedCREST, gastrointestinal dysmotility and spasm, and abnormalities in thecentral, peripheral and autonomic nervous system.

The major symptoms or manifestations of scleroderma and in particular ofsystemic sclerosis are inappropriate excessive collagen synthesis anddeposition, endothelial dysfunction, vasospasm, collapse andobliteration of vessels by fibrosis.

For example, a compound having the structure as shown in Formula I canbe ajulemic acid. In particular, applicants have discovered thatadministration of ultrapure ajulemic acid is effective in treatingtissue fibrosis of the lung and skin, as demonstrated using awell-established animal model of scleroderma without any of theCB1-mediated behavioral side effects.

A therapeutically effective amount of the present compound (e.g.,ultrapure ajulemic acid) may lower the level of pain experienced by asubject. In one embodiment, the level of pain experienced by a patientcan be assessed by use of a visual analog scale (VAS) or a Likert-typescale. A VAS is a straight line with one end of the line representing nopain and the other end of the line representing the worst imaginablepain. Patients are asked to mark on the line where they considered theirpain to be at each time point, and the length from no pain to the markcan be related to the length of the full scale. A Likert-type scale is arating scale, usually in the range of 1 to 5, based on degrees ofagreement or disagreement to statements. A similar type of scale,although based on an 11 point scale (ranging from 0 to 10) can also beused. Such pain scales can be applied to visualize an alteration of thelevel of pain a patient experiences during treatment, e.g., a reductionof the level of pain a patient or a population of patients experiencesbefore and after initiation of a pain therapy. U.S. Pat. No. 7,413,748.For example, the pain may be reduced by at least about 1 point, at leastabout 2 points, at least about 3 points, at least about 4 points, atleast about 5 points, at least about 6 points, at least about 7 points,or at least about 8 points on an 11-point pain scale. The level of painmay also be assessed by other suitable methods.

A therapeutically effective amount of the present compound (e.g.,ultrapure ajulemic acid) may be used to treat or prevent fibrosis.Fibrosis may be assessed using in vitro or in vivo models. In oneembodiment, in vitro fibrosis can be assayed by measuring the amount ofextracellular matrix protein production in response to TGF-beta, PDGF,CTGF, or other pro-fibrotic factors or through the presence of markersof fibroblast activation. Common endpoints include measurement ofcollagen, fibronectin, and actin. In another embodiment, in vivofibrosis is measured by the degree of extracellular matrix production ina particular tissue. In vivo models of fibrosis include chemicallyinduced models in which an external fibrosis mediator such as bleomycin,HOCl, CCl₄ or alcohol is used to induce liver, kidney, skin or lungfibrosis. Genetic models of fibrosis are also commonly used includinganimals which overexpress TGF-beta, PDGF, osteopontin and interleukins,plus the tight skin (tsk) mouse model. Fibrosis may also be assessed byother suitable methods.

A therapeutically effective amount of the present compound (e.g.,ultrapure ajulemic acid) may be used to treat or prevent inflammation.Inflammation may be assessed using in vitro or in vivo models. In oneembodiment, in vitro inflammation can be assayed by measuring thechemotaxis and activation state of inflammatory cells. In anotherembodiment, inflammation can be measured by examining the production ofspecific inflammatory mediators such as interleukins, cytokines andeicosanoids mediators. In yet another embodiment, in vivo inflammationis measured by swelling and edema of a localized tissue or migration ofleukocytes. Animal models of inflammation may use a stimulus such asphorbol esters, arachidonic acid, platelet activating factor, zymosan,LPS, thioglycollate or other agents to elicit inflammation in tissuessuch as ear, paw, skin or peritonium. Inflammation may also be measuredby organ function such as in the lung or kidneys and by the productionof pro-inflammatory factors. Inflammation may also be assessed by othersuitable methods.

Methods of Treatment

The compounds and compositions described herein can be administered tocells in culture, e.g. in vitro or ex vivo, or to a subject, e.g., invivo, to treat, prevent, and/or diagnose a variety ofconditions/diseases as discussed above and further described below.

The term “treat” or “treatment” includes administration of a compound,e.g., by any route, e.g., orally, to a subject. The compound can beadministered alone or in combination with, a second compound. Treatmentsmay be sequential, with the present compound being administered beforeor after the administration of other agents. Alternatively, agents maybe administered concurrently. The subject, e.g., a patient, can be onehaving a disorder (e.g., a disorder as described herein), a symptom of adisorder, or a predisposition toward a disorder. Treatment is notlimited to curing or complete healing, but can result in one or more ofalleviating, relieving, altering, partially remedying, ameliorating,improving or affecting the disorder, reducing one or more symptoms ofthe disorder or the predisposition toward the disorder. In an embodimentthe treatment (at least partially) alleviates or relieves fibrosis. Inone embodiment, the treatment reduces at least one symptom of thedisorder or delays onset of at least one symptom of the disorder. Theeffect is beyond what is seen in the absence of treatment.

The compound effective to treat a disorder refers to an amount of thecompound which is effective, upon single or multiple dose administrationto a subject, to achieve treatment. The degree of treatment with atherapeutically effective amount encompasses any improvement or cure ofa disease as measured by standard clinically relevant criteria.

The amount of a compound effective to prevent a disorder, or “aprophylactically effective amount” of the compound refers to an amounteffective, upon single- or multiple-dose administration to the subject,in preventing or delaying the occurrence of the onset or recurrence of adisorder or a symptom of the disorder.

Subjects that can be treated with the compounds and methods of thepresent invention include both human and non-human animals. Exemplaryhuman subjects include a human patient having a disorder, e.g., adisorder described herein or a normal subject. The term “non-humananimals” of the invention includes all vertebrates, e.g., non-mammals(such as chickens, amphibians, reptiles) and mammals, such as non-humanprimates, domesticated and/or agriculturally useful animals, e.g.,sheep, dog, cat, cow, pig, etc. In an embodiment the animal is otherthan a rodent, e.g., a rat or mouse, or a non-human primate.

Titration of a Subject

Treatment of subjects can be optimized by titrating the subject, forexample, such that treatment can be initiated with sub-optimal orno-effect doses of each compound and increased to determine the optimaldose of ultrapure ajulemic acid for the treatment and/or prevention offibrotic or inflammatory diseases in the subject.

Treating a subject with ultrapure ajulemic acid can cause side effectssuch as dizziness, dry mouth, headache, nausea, pallor, somnolence, andvomiting.

The side effects can be modulated to some extent by starting at a lowdose and slowly titrating the dose upward, e.g., during the course oftreatment, for example over the course of weeks, months or years.

In one embodiment, a subject is titrated to minimize the adverse eventsand achieve a therapeutic level of the appropriate dosage form ofultrapure ajulemic acid.

Pharmaceutical Compositions

Various dosage forms of the present ultrapure compounds (e.g., ultrapureajulemic acid) can be used in the methods of the present invention forpreventing and/or treating various conditions with a better safety andtolerability profile than the prior ajulemic acid. In certainembodiments, the dosage form is an oral dosage form such as a tablet orcapsule or enteric coated tablet or osmotic release capsule or uniquecombination of excipients. In other embodiments, the dosage form is aliquid, topical patch, gel, ointment, cream, aerosol, or inhaledformulation.

The present compositions may be formulated to deliver over a 24-hourperiod from about 0.5 mg to about 240 mg, from about 5 mg to about 180mg, or from about 10 mg to about 120 mg of the present ultrapurecompound (e.g., ultrapure ajulemic acid).

In further embodiments, the dosage form includes an additional agent oris provided together with a second dosage form, which includes theadditional agent. Exemplary additional agents include an analgesic agentsuch as an NSAID or opiate, an anti-inflammatory agent or a naturalagent such as a triglyceride containing unsaturated fatty acid, orisolated pure fatty acids such as eicosapentaenoic acid (EPA),dihomogamma linolenic acid (DGLA), docosahexaenoic acid (DHA) andothers. In additional embodiments, the dosage form comprises a capsulewherein the capsule contains a mixture of materials to provide a desiredsustained release formulation.

The dosage forms can include a tablet coated with a semipermeablecoating. In certain embodiments, the tablet comprises two layers, alayer containing ultrapure ajulemic acid and a second layer referred toas a “push” layer. The semi-permeable coating is used to allow a fluid(e.g., water) to enter the tablet and erode a layer or layers. Incertain embodiments, this sustained release dosage form furthercomprises a laser hole drilled in the center of the coated tablet. Theajulemic acid or other (3R,4R)-Δ8-tetrahydrocannabinol-11-oic acidcontaining layer comprises ajulemic acid or another(3R,4R)-Δ8-tetrahydrocannabinol-11-oic acid, a disintegrant, a viscosityenhancing agent, a binding agent and an osmotic agent. The push layercomprises a disintegrant, a binding agent, an osmotic agent and aviscosity enhancing agent.

In another aspect, the invention features a dosage form of ultrapureajulemic acid that is a controlled release dosage form, which providescontrolled release of ultrapure ajulemic acid.

In further embodiments, the dosage form comprises a tablet comprising abiocompatible matrix and ultrapure ajulemic acid. The sustained releasedosage form may also comprise a hard-shell capsule containingbio-polymer microspheres that contains the therapeutically active agent.The biocompatible matrix and bio-polymer microspheres each contain poresfor drug release and delivery. These pores are formed by mixing thebiocompatible matrix of bio-polymer microsphere with a pore formingagent. Each biocompatible matrix or bio-polymer microsphere is made upof a biocompatible polymer or mixture of biocompatible polymers. Thematrix and microspheres can be formed by dissolving the biocompatiblepolymer and active agent (compound described herein) in a solvent andadding a pore-forming agent (e.g., a volatile salt). Evaporation of thesolvent and pore forming agent provides a matrix or microspherecontaining the active compound. In additional embodiments, the sustainedrelease dosage form comprises a tablet, wherein the tablet containsultrapure ajulemic acid and one or more polymers and wherein the tabletcan be prepared by compressing the ultrapure ajulemic acid and one ormore polymers. In some embodiments, the one or more polymers maycomprise a hygroscopic polymer formulated with ultrapure ajulemic acid.Upon exposure to moisture, the tablet dissolves and swells. Thisswelling allows the sustained release dosage form to remain in the upperGI tract. The swelling rate of the polymer mixture can be varied usingdifferent grades of polyethylene oxide.

In other embodiments, the sustained release dosage form comprises acapsule further comprising particle cores coated with a suspension ofactive agent and a binding agent which is subsequently coated with apolymer. The polymer may be a rate-controlling polymer. In general, thedelivery rate of the rate-controlling polymer is determined by the rateat which the active agent is dissolved.

Various dosage forms of ultrapure ajulemic acid can be administered to asubject. Exemplary dosage forms include oral dosage forms (e.g., atablet or capsule), topical dosage forms such as a topical patch, gels,and ointments, ophthalmic dosage forms such as drops or liquidformulations, interstitial dosage forms such as liquid formulations, andinhaled dosage forms such as inhalers, nebulizers, aerosols and sprays.

In certain embodiments, the ultrapure ajulemic acid is formulated into adosage form wherein a single dosage is from about 0.5 mg to about 120 mgonce daily or from about 0.15 mg to about 40 mg up to 3 times daily.

In other embodiments, the ultrapure ajulemic acid is formulated into adosage form wherein a single dosage is from about 0.01 to about 1.5mg/kg weight of the subject. In further embodiments, the dosage form isadministered up to 3 times daily and from about 0.003 to about 0.5 mg/kgweight of the subject.

As used herein, the term “therapeutically effective amount” is an amountsufficient to treat a specified disorder or disease or alternatively toobtain a pharmacological response treating a disorder or disease.Methods of determining the most effective means and dosage ofadministration can vary with the composition used for therapy, thepurpose of the therapy, the target cell being treated, and the subjectbeing treated. Treatment dosages generally may be titrated to optimizesafety and efficacy. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician. Suitable dosage formulations and methods of administering theagents can be readily determined by those of skill in the art. Forexample, the composition may be administered at about 0.01 mg/kg toabout 200 mg/kg, about 0.1 mg/kg to about 100 mg/kg, or about 0.5 mg/kgto about 50 mg/kg. When the compounds described herein areco-administered with another agent or therapy, the effective amount maybe less than when the agent is used alone.

In an embodiment, one or more of the therapeutic agents that can be usedin the methods of the present invention for preventing and/or treatingconditions discussed above are formulated with a pharmaceuticallyacceptable carrier, vehicle or adjuvant. The term “pharmaceuticallyacceptable carrier, vehicle or adjuvant” refers to a carrier, vehicle oradjuvant that may be administered to a subject, together with thepresent compounds, and which does not destroy the pharmacologicalactivity thereof and is nontoxic when administered in doses sufficientto deliver a therapeutic amount of the compound.

The compound may be formulated as a salt such as a pharmaceuticallyacceptable salt form, which includes, but are not limited to, acidaddition salts formed with inorganic acids (e.g. hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and thelike), and salts formed with organic acids such as, but not limited to,acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid,fumaric acid, maleic acid, ascorbic acid, benzoic acid, tannic acid,pamoic acid, alginic acid, polyglutamic, acid, naphthalene sulfonicacid, naphthalene disulfonic acid, and polygalacturonic acid.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Suitable pharmaceutically-acceptable baseaddition salts include metallic salts, such as salts made from aluminum,calcium, lithium, magnesium, potassium, sodium and zinc, or salts madefrom organic bases including primary, secondary and tertiary amines,substituted amines including cyclic amines, such as caffeine, arginine,diethylamine, N-ethyl piperidine, histidine, glucamine, isopropylamine,lysine, morpholine, N-ethyl morpholine, piperazine, piperidine,triethylamine, trimethylamine. All of these salts may be prepared byconventional means from the corresponding compound of the invention byreacting, for example, the appropriate acid or base with the compound ofthe invention. Handbook of Pharmaceutical Salts: Properties, and Use (P.H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002) [1].

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the dosage forms of this invention include, but are not limitedto, ion exchangers, alumina, aluminum stearate, lecithin,self-emulsifying drug delivery systems (SEDDS) such as d-E-tocopherolpolyethylene-glycol 1000 succinate; surfactants used in pharmaceuticaldosage forms such as Tweens or other similar polymeric deliverymatrices; serum proteins such as human serum albumin; buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts; orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxmethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat. Cyclodextrins such as alpha, beta andγ-cyclodextrin, or chemically modified derivatives such ashydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-betacyclodextrins, or other solubilized derivatives may also beadvantageously used to enhance delivery of compounds of the formulaedescribed herein that can be used in the methods of the presentinvention for preventing and/or treating fibrotic conditions. Additionalsuitable excipients may be found in Handbook of PharmaceuticalExcipients, R. C. Rowe, et. al., Pharmaceutical Press, 2009 [9]. Incertain embodiments, unit dosage formulations are compounded forimmediate release, though unit dosage formulations compounded fordelayed or prolonged release of one or both agents are also disclosed.

In one embodiment, the therapeutic agents that can be used in thepresent methods are formulated in a single unit dose such that theagents are released from the dosage at different times.

In another embodiment, for example, where one or more of the therapeuticagents is administered once or twice per day, the agent is formulated toprovide extended release. For example, the agent is formulated with anenteric coating. In an alternative embodiment, the agent is formulatedusing a biphasic controlled release delivery system, thereby providingprolonged gastric residence. For example, in some embodiments, thedelivery system includes (1) an inner solid particulate phase formed ofsubstantially uniform granules containing a pharmaceutical having a highwater solubility, and one or more hydrophilic polymers, one or morehydrophobic polymers and/or one or more hydrophobic materials such asone or more waxes, fatty alcohols and/or fatty acid esters, and (2) anouter solid continuous phase in which the above granules of inner solidparticulate phase are embedded and dispersed throughout, the outer solidcontinuous phase including one or more hydrophobic polymers, one or morehydrophobic polymers and/or one or more hydrophobic materials such asone or more waxes, fatty alcohols and/or fatty acid esters, which may becompressed into tablets or filled into capsules. In some embodiments,the agent is incorporated into polymeric matrices comprised ofhydrophilic polymers that swell upon imbibition of water to a size thatis large enough to promote retention of the dosage form in the stomachduring the fed mode.

The ultrapure ajulemic acid in the formulation may be formulated as acombination of fast-acting and controlled release forms. For example,the ultrapure ajulemic acid is formulated with a single releaseproperty. For example, it is not present in a modified release form,e.g., a controlled release form.

The present compositions may be taken just prior to or with each ofthree meals, each of two major meals, or one meal. In other embodiments,a composition disclosed herein can be administered once a day or twice aday and need not be administered just before or with a meal.

The dosage forms of this invention that can be used in the presentmethods may be administered orally, parentally, by inhalation spray,topically, rectally, interstitially, nasally, buccally, vaginally or viaan implanted reservoir, preferably by oral administration oradministration by injection. The pharmaceutical compositions of thisinvention may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles. In somecases, the pH of the formulation may be adjusted with pharmaceuticallyacceptable acids, bases or buffers to enhance the stability of theformulated compound or its delivery form. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional and intracranial injection orinfusion techniques.

The dosage forms that can be used in the present methods may be in theform of a sterile injectable preparation, for example, as a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to techniques known in the art using suitabledispersing or wetting agents (such as, for example, Tween 80) andsuspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a nontoxic parenterallyacceptable diluent or solvent, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are mannitol, water, Ringer's solution and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose, any blandfixed oil may be employed including synthetic mono- or diglycerides.Fatty acids, such as oleic acid and its glyceride derivatives are usefulin the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,or carboxymethyl cellulose or similar dispersing agents which arecommonly used in the formulation of pharmaceutically acceptable dosageforms such as emulsions and or suspensions. Other commonly usedsurfactants such as Tweens or Spans and/or other similar emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

The present compounds or compositions may be administered orally, forexample as a component in a dosage form. The dosage forms may containany conventional non-toxic pharmaceutically-acceptable carriers,adjuvants or vehicles. In some cases, the pH of the formulation may beadjusted with pharmaceutically acceptable acids, bases or buffers toenhance the stability of the formulated compound or its delivery form.

The dosage forms of this invention may be orally administered in anyorally acceptable dosage form including, but not limited to, capsules,tablets, emulsions and aqueous suspensions, dispersions and solutions.In the case of tablets for oral use, carriers that are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch. Whenaqueous suspensions and/or emulsions are administered orally, the activeingredient may be suspended or dissolved in an oily phase is combinedwith emulsifying and/or suspending agents. If desired, certainsweetening and/or flavoring and/or coloring agents may be added.

The dosage forms of this invention may also be administered in the formof suppositories for rectal administration. These compositions can beprepared by mixing a compound of this invention that can be used in themethods of the present invention for preventing and/or treating fibroticconditions with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

Ophthalmic administration of the dosage forms of this invention isuseful when the desired treatment involves areas or organs readilyaccessible by ophthalmic application. Ophthalmic administration, thecomposition could be applied by instillation of a cream, an ointment, ora liquid drop preparation in the conjunctival sac.

Topical administration of the dosage forms of this invention is usefulwhen the desired treatment involves areas or organs readily accessibleby topical application. For application topically to the skin, thedosage form should be formulated with a suitable ointment containing theactive components suspended or dissolved in a carrier. Carriers fortopical administration of the compounds of this invention include, butare not limited to, mineral oil, liquid petroleum, white petroleum,propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifyingwax and water. Alternatively, the pharmaceutical composition that can beused in the methods of the present invention can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier with suitable emulsifying agents. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water. The pharmaceuticalcompositions of this invention may also be topically applied to thelower intestinal tract by rectal suppository formulation or in asuitable enema formulation. Topically-transdermal patches are alsoincluded in this invention.

The dosage forms of this invention that can be used in the methods ofthe present invention may be administered by nasal aerosol orinhalation. Such compositions are prepared according to techniqueswell-known in the art of pharmaceutical formulation and may be preparedas solutions in saline, employing benzyl alcohol or other suitablepreservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

When the dosage forms of this invention comprise a combination of acompound of the formulae described herein and one or more additionaltherapeutic or prophylactic agents, both the compound and the additionalagent should be present at dosage levels of between about 1 to 100%, andmore preferably between about 5 to 95% of the dosage normallyadministered in a monotherapy regimen. The additional agents may beadministered separately, as part of a multiple dose regimen, from thecompounds of this invention. Alternatively, those agents may be part ofa single dosage form, mixed together with the compounds of thisinvention in a single composition.

In certain embodiments, the dosage form that can be used in the methodsof the present invention comprises a capsule wherein the capsulecomprises a mixture of material to provide the desired sustainedrelease.

In other embodiments, the dosage form that can be used in the methods ofthe present invention comprises a tablet coated with a semipermeablecoating. In certain embodiments, the tablet comprises two layers, alayer containing ultrapure ajulemic acid and a second layer referred toas a “push” layer. The semi-permeable coating is used to allow a fluid(e.g., water) to enter the tablet and erode a layer or layers. Incertain embodiments, the sustained release dosage form further comprisesa laser hole drilled in the center of the coated tablet. The ultrapureajulemic acid containing layer comprises ultrapure ajulemic acid, adisintegrant, a viscosity enhancing agent, a binding agent and anosmotic agent. The push layer comprises a disintegrant, a binding agent,an osmotic agent and a viscosity-enhancing agent.

In further embodiments, the dosage form that can be used in the methodsof the present invention comprises a tablet comprising a biocompatiblematrix and an ultrapure ajulemic acid. The sustained release dosage formmay also comprise a hard-shell capsule containing bio-polymermicrospheres that contains the therapeutically active agent. Thebiocompatible matrix and bio-polymer microspheres each contain pores fordrug release and delivery. These pores are formed by mixing thebiocompatible matrix or bio-polymer microsphere with a pore formingagent. Each biocompatible matrix of bio-polymer microsphere is made upof a biocompatible polymer or mixture of biocompatible polymers. Thematrix and microspheres can be formed by dissolving the biocompatiblepolymer and active agent (compound described herein) in a solvent andadding a pore forming agent (e.g., a volatile salt). Evaporation of thesolvent and pore forming agent provides a matrix or microspherecontaining the active compound.

The sustained release dosage form that can be used in the methods of thepresent invention comprises a tablet, wherein the tablet containsultrapure ajulemic acid and one or more polymers and wherein the tabletcan be prepared by compressing the ultrapure ajulemic acid and one ormore polymers. In some embodiments, the one or more polymers maycomprise a hygroscopic polymer formulated with the ultrapure ajulemicacid active agent (i.e., a compound described herein). Upon exposure tomoisture, the tablet dissolves and swells. This swelling allows thesustained release dosage form to remain in the upper GI tract. Theswelling rate of the polymer mixture can be varied using differentgrades of polyethylene oxide.

In other embodiments, the sustained release dosage form that can be usedin the methods of the present invention comprises a capsule furthercomprising particle cores coated with a suspension of active agent and abinding agent which is subsequently coated with a polymer. The polymermay be a rate-controlling polymer. In general, the delivery rate of therate-controlling polymer is determined by the rate at which the activeagent is dissolved.

Non-limiting examples of capsules include but are not limited to gelatincapsules, HPMC, hard shell, soft shell, or any other suitable capsulefor holding a sustained release mixture.

The solvents used in the above sustained release dosage forms include,but are not limited to ethyl acetate, triacetin, dimethyl sulfoxide(DIV1S0), propylene carbonate, N-methylpyrrolidone (NMP), ethyl alcohol,benzyl alcohol, glycofurol, alpha-tocopherol, Miglyol 810, isopropylalcohol, diethyl phthalate, polyethylene glycol 400 (PEG 400), triethylcitrate, and benzyl benzoate.

The viscosity modifiers used in the above sustained release dosage formsinclude, but are not limited to caprylic/capric triglyceride (Migliol810), isopropyl myristate (IPM), ethyl oleate, triethyl citrate,dimethyl phthalate, benzyl benzoate and various grades of polyethyleneoxide. The high viscosity liquid carrier used in the above sustainedrelease dosage forms include, but are not limited to sucrose acetateisobutyrate (SAIB) and cellulose acetate butyrate (CAB) 381-20.

Non-limiting examples of materials that make up preferred semi-permeablelayers include, but are not limited to cellulosic polymers such ascellulose acetate, cellulose acylate, cellulose diacylate, cellulosetriacylate, cellulose diacetate, cellulose triacetate or any mixturesthereof; ethylene vinyl acetate copolymers, polyethylene, copolymers ofethylene, polyolefins including ethylene oxide copolymers (e.g.,Engage®—Dupont Dow Elastomers), polyamides, cellulosic materials,polyurethanes, polyether blocked amides, and copolymers (e.g., PEBAX®,cellulosic acetate butyrate and polyvinyl acetate). Non-limitingexamples of disintegrants that may be employed in the above sustainedrelease dosage forms include but are not limited to croscarmellosesodium, crospovidone, sodium alginate or similar excipients.

Non-limiting examples of binding agents that may be employed in theabove sustained release dosage forms include but are not limited tohydroxyalkylcellulose, a hydroxyalkylalkylcellulose, or apolyvinylpyrrolidone.

Non-limiting examples of osmotic agents that may be employed in theabove sustained release dosage forms include but are not limited to,sorbitol, mannitol, sodium chloride, or other salts. Non-limitingexamples of biocompatible polymers employed in the above sustainedrelease dosage forms include but are not limited to poly(hydroxylacids), polyanhydrides, polyorthoesters, polyamides, polycarbonates,polyelkylenes, polyelkylene glycols, polyalkylene oxides, polyalkyleneterepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters,polyvinyl halides, polyvinylpyrrolidone, polysiloxanes, poly(vinylalcohols), poly (vinyl acetate), polystyrene, polyurethanes andco-polymers thereof, synthetic celluloses, polyacrylic acids,poly(butyric acid), poly(valeric acid), andpoly(lactide-co-caprolactone), ethylene vinyl acetate, copolymers andblends thereof.

Non-limiting examples of hygroscopic polymers that may be employed inthe above sustained release dosage forms include but are not limited topolyethylene oxide (e.g., Polyox® with MWs from 4,000,000 to10,000,000), cellulose hydroxymethyl cellulose, hydroxyethyl-cellulose,crosslinked polyacrylic acids and xanthum gum.

Non-limiting examples of rate-controlling polymers the may be employedin the above sustained release dosage forms includes but is not limitedto polymeric acrylate, methacrylatelacquer or mixtures thereof,polymeric acrylate lacquer, methacrylate lacquer, an acrylic resincomprising a copolymer of acrylic and methacrylic acid esters or anammonium methacrylate lacquer with a plasticizer.

Kits

A dosage form described herein may be provided in a kit. The kitincludes (a) a compound used in a method described herein, and,optionally (b) informational material. The informational material can bedescriptive, instructional, marketing or other material that relates tothe methods described herein and/or the use of the dosage form for themethods described herein.

The informational material of the kits is not limited in its form. Inone embodiment, the informational material can include information aboutproduction of the compound, molecular weight of the compound,concentration, date of expiration, batch or production site information,and so forth. In one embodiment, the informational material relates tomethods for administering the compound.

In one embodiment, the informational material can include instructionsto use a compound described herein in a suitable manner to perform themethods described herein, e.g., carry out a reaction to produce acompound described herein.

The informational material of the kits is not limited in its form. Inmany cases, the informational material, e.g., instructions, is providedin printed matter, e.g., a printed text, drawing, and/or photograph,e.g., a label or printed sheet. However, the informational material canalso be provided in other formats, such as Braille, computer readablematerial, video recording, or audio recording. In another embodiment,the informational material of the kit is contact information, e.g., aphysical address, email address, website, or telephone number, where auser of the kit can obtain substantive information about a compounddescribed herein and/or its use in the methods described herein. Ofcourse, the informational material can also be provided in anycombination of formats.

In addition to a dosage form described herein, the composition of thekit can include other ingredients, such as a solvent or buffer, astabilizer, a preservative, a flavoring agent (e.g., a bitter antagonistor a sweetener), a fragrance, a dye or coloring agent, for example, totint or color one or more components in the kit, or other cosmeticingredient, and/or a second agent for treating a condition or disorderdescribed herein. Alternatively, the other ingredients can be includedin the kit, but in different compositions or containers than a compounddescribed herein. In such embodiments, the kit can include instructionsfor admixing a compound described herein and the other ingredients, orfor using a compound described herein together with the otheringredients.

In some embodiments, the components of the kit are stored under inertconditions (e.g., under Nitrogen or another inert gas such as Argon). Insome embodiments, the components of the kit are stored under anhydrousconditions (e.g., with a desiccant). In some embodiments, the componentsare stored in a light blocking container such as an amber vial.

A dosage form described herein can be provided in any form, e.g.,liquid, dried or lyophilized form. It is preferred that a compounddescribed herein be substantially pure and/or sterile. When a compounddescribed herein is provided in a liquid solution, the liquid solutionpreferably is an aqueous solution, with a sterile aqueous solution beingpreferred. When a compound described herein is provided as a dried form,reconstitution generally is by the addition of a suitable solvent. Thesolvent, e.g., sterile water or buffer, can optionally be provided inthe kit.

The kit can include one or more containers for the compositioncontaining a dosage form described herein. In some embodiments, the kitcontains separate containers, dividers or compartments for thecomposition and informational material. For example, the composition canbe contained in a bottle, vial, or syringe, and the informationalmaterial can be contained in a plastic sleeve or packet. In otherembodiments, the separate elements of the kit are contained within asingle, undivided container. For example, the dosage form is containedin a bottle, vial or syringe that has attached thereto the informationalmaterial in the form of a label. In some embodiments, the kit includes aplurality (e.g., a pack) of individual containers, each containing oneor more unit dosage forms (e.g., a dosage form described herein) of acompound described herein. For example, the kit includes a plurality ofsyringes, ampules, foil packets, or blister packs, each containing asingle unit dose of a dosage form described herein.

The containers of the kits can be air tight, waterproof (e.g.,impermeable to changes in moisture or evaporation), and/or light-tight.

The kit optionally includes a device suitable for use of the dosageform, e.g., a syringe, pipette, forceps, measured spoon, swab (e.g., acotton swab or wooden swab), or any such device.

Thus, specific compositions and ultrapure tetrahydrocannabinol-11-oicacids have been disclosed. A number of embodiments of the invention havebeen described. Nevertheless, it will be understood that variousmodifications may be made without departing from the spirit and scope ofthe invention. Accordingly, other embodiments are within the scope ofthe following claim. It should be apparent, however, to those skilled inthe art that many more modifications besides those already described arepossible without departing from the inventive concepts herein. Theinventive subject matter, therefore, is not to be restricted except inthe spirit of the disclosure. All patents, patent publications andpublications mentioned herein are incorporated herein by reference intheir entirety to disclose and describe the methods and/or materials inconnection with which the publications are cited. The publicationsdiscussed herein are provided solely for their disclosure prior to thefiling date of the present application. Nothing herein is to beconstrued as an admission that the present invention is not entitled toantedate such publication by virtue of prior invention.

EXAMPLES

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1 Evaluation of Ultrapure Aja in Regards to CB1 and CB2

Ultrapure ajulemic acid (JBT-101) was synthesized and its binding to CB1and CB2 receptors was compared to that of previous preparations.

With respect to ultrapure AJA, there is a significant difference in itsK_(i) in regards to CB1 and CB2. In one particular embodiment, as shownin FIG. 9, the binding affinity of ultrapure AJA for CB2 is about 10× to20× greater than the binding affinity for CB1. For comparison, FIG. 9shows various other K_(i) and K_(i)(CB1)/K_(i)(CB2) ratios for variousother cannabinoids and other previous synthetically produced AJA [10,11].

Example 2

Direct Comparisons of AJA Vs. THC in the Same Studies

-   -   1. Binding-CB1/CB2* CB2 binding is a highly desirable property        since it appears to mediate anti-inflammatory and anti-fibrotic        effects without psychotropic action.    -   2. Ring test-Cataleptic response generally accepted as a        psychotropic effect mediated by CB1 agonist activity at the        level of the brain. Each compound was tested at various        therapeutic doses. [11]    -   3. In vivo Cancer. AJA shows a small but significant inhibition        of tumor growth greater than that produced by THC. The doses        chosen were in the anti-inflammatory range [12].    -   4. PK Data. While it shows some cannabinoid-like CNS activity at        higher doses, it exhibits a superior therapeutic index compared        to other cannabinoid compounds, which may reflect a relatively        reduced CNS penetration [3]. Moreover, the pharmacokinetic        analysis indicates that although there is some brain penetration        in the rat, it is restricted to a degree, with peak levels in        the brain, measured at the peak pharmacodynamic time points,        reaching only 25-30% of those seen in the plasma. This contrasts        with the profile observed with WIN55,212-2 and THC which show a        significantly higher relative brain penetration, with brain        levels reaching 100-190% of those seen in the plasma. These data        complement the recent findings in man in which AJA was found to        reduce pain scores in neuropathic pain patients in the absence        of cannabis-like psychotropic adverse events [13].

Example 3 Study of Ultrapure AJA's Activity on CB1 and CB2 Receptors

Ultrapure ajulemic acid (JBT-101) was evaluated for its pharmacologicaleffects in mice, as well as for functional in vitro activation of CB1and CB2 cannabinoid receptors.

Specifically, JBT-101 was assessed for its ability to stimulate[35S]GTPγS turnover in CB1 and CB2 receptors in vitro. The compound wasalso assessed for its antinociceptive effects in a hot plate assay andfor cataleptic effects in a ring immobility test, both in female CD-1mice. Rectal temperature was also measured in these mice.

Introduction of Methods Used Hot Plate Test for Antinociception

The hot-plate test was used to measure the analgesic activity ofultrapure AJA and other pharmacologic agents based on the reaction timeof mice to lick their forepaws and/or jump after being placed on analuminum hot plate heated to, and maintained at, 54° C.-56° C. Kitchen Iand Green P G, Differential Effects of DFP Poisoning and Its Treatmenton Opioid Antinociception in the Mouse, Life Sci. 33:669-672 (1983).This test has been shown to measure CB1 agonist activity.

An aluminum surface was maintained at 55° C.±1° C. by circulating waterthrough the passages in the metal. A clear plastic cylinder, 18 cm indiameter and 26 cm high, was placed on the surface to prevent escape.The end point was taken as the time when the mouse either performed ahind paw lick or jumped off the surface; in no case were the animalskept more than 30 seconds on the plate. Mice were never used more thanone time; control values and test values were measured, e.g., 3 hoursapart. Ultrapure AJA and other test compounds were administered orallyabout ninety (90) minutes before the hot plate test. The percent changein response time (latency) was calculated by comparing the mean of thecontrol values with the mean of the test values and statisticalsignificance determined by a paired t test.

A dose response was conducted for ultrapure AJA at doses from 0.05 to 56mg/kg. The ultrapure AJA required much higher doses than the AJA fromU.S. Pat. No. 5,338,753 to see analgesia.

Measurement of Cataleptic Effects

The cataleptic response was measured using the ring test described byPertwee (Pertwee R G, The Ring Test. A Quantitative Method of Assessingthe Cataleptic Effect of Cannabis in Mice, Br. J. Pharmacol. 46:753-763(1972)). Mice were placed on a horizontal wire ring 5.5 cm in diameterattached to a 16 cm vertical rod. The hind paws and fore paws wereplaced at opposite sides of the ring. The ambient temperature wasmaintained at 30° C. and the environment was free of auditory stimuliand bright lights. The response was calculated as the fraction of timethe mouse was immobile over a five (5) minute test period.

A dose response was conducted for ultrapure AJA to oral doses of 0.05 to56 mg/kg. The ultrapure AJA required much higher doses then the AJA (3a)from the U.S. Pat. No. 5,338,753 to see catalepsy.

GTP-Gamma-S Assay

When the CB1 or CB2 receptor is activated by an agonist, the affinity ofthe G protein alpha subunit increases with respect to GTP vs. GDP. As aconsequence, GDP is displaced from the G protein and GTP or GTPγS binds.If a radioactive label, such as [³⁵S], is attached to the GTPγSmolecule, then the formation of the G protein/[³⁵S]GTPγS complex can bedirectly measured using liquid scintillation spectrophotometry. Weilandet al., (1994) Methods Enzymol 237:3-13. Griffin et al., PET285:553-560, 1998.

A GTP-gamma-S assay was used to study the functional activity of AJA onhuman CB1 and CB2 receptors to further determine the selectivity ofultrapure AJA for the CB2 receptor. As shown in FIG. 13, the potency ofultrapure AJA in the GTP-gamma-S assay was ˜10× better in the CB2 assaythan in the CB1 assay which further supporting the improved selectivityof ultrapure AJA for CB2 vs CB1.

Experimental Details Preparation of Stock Solutions for In VitroFunctional Assay

For in vitro functional assays, the JBT-101 stock solution was preparedin ethanol or DMSO.

Preparation of Solutions for In Vivo Testing

Δ⁹-THC [National Institute on Drug Abuse (NIDA), Rockville, Md.],indomethacin (Sigma-Aldrich, St. Louis, Mo.), and JBT-101 were dissolvedin a vehicle of peanut or safflower oil (food grade). Compounds wereadministered at a volume of 20 μl/kg via oral gavage.

In Vitro Functional Assay at Cannabinoid Receptors Materials and Methods

The CB1 and CB2 receptor assays involve membrane preparations purchasedfrom Perkin Elmer (Waltham, Mass.) isolated from a HEK-293 expressionsystem. G-protein coupled signal transduction (GTP-7-[35S]) assays oftest compounds were conducted in an incubation mixture consisting of atest compound (250 nM-1 mM), GDP (20 μM), GTP-7-[35S] (100 μM), and thehCB₁ and hCB₂ membrane preparations (0.4 μM) in a total volume of 0.4 mLof assay buffer (50 mM TRIS-HCl, pH 7.4, 1 mM EDTA, 100 mM NaCl, 5 mMMgCl2, 0.5% (w/v) BSA). Nonspecific binding was determined in thepresence of 100 μM unlabeled GTP-γ-S, and basal binding was determinedin the absence of drug. Duplicate samples were incubated for 1 h at 30°C., and the bound complex was filtered from the reaction mixture asdescribed previously and counted in a liquid scintillation counter.Specific binding was calculated by subtracting nonspecific binding fromtotal binding and dividing by the total basal binding minus nonspecificbinding. Data were plotted and analyzed with GraphPad Prism (GraphPadSoftware, Inc., San Diego, Calif.).

In Vitro Assays: Results and Discussion

CP55940 (positive control) stimulated GTP-7-35S turnover through boththe hCB1 receptor and the hCB2 receptor at nM concentrations (Table 2;EC50=9.99±2.5 nM for CB1 and EC50=3.96±1.3 nM for CB2). These resultsindicate that CP55,940 acts as agonists at these G-protein coupledreceptor sites (FIG. 13, upper panel). JBT-101 also stimulated GTP-7-35Sturnover through both cannabinoid receptors, but did so with much lesspotency (FIG. 13, lower panel). The EC50 for JBT-101 at the CB1 receptorwas 9209±2042 nM whereas the EC50 at the CB2 receptor was 1020±92 nM(Table 2). The 9-fold difference in potency for activation of CB2 versusCB1 receptors is consistent with the 12-fold selectivity of thiscompound for binding CB2 receptors, suggesting that JBT-101 wouldactivate CB2 receptors at doses that are not active at CB1 receptors.

TABLE 2 GTP-γ-35S turnover in hCB1 and hCB2 receptors. Com- Mean poundRep 1 Rep 2 Rep 3 Rep 4 (±SEM) hCB₁ EC₅₀ (nM) CP55, 940 6.989 6.2949.529 17.14 9.99 (2.5) JBT-101 13694 5830 11647 5664 9209 (2042) hCB₂EC₅₀ (nM) CP55, 940 2.81 0.69 5.91 6.41 3.96 (1.3) JBT-101 930 815 12321101 1020 (92)

Mouse In Vivo Tests Subjects

Female CD-1 mice (20-25 g), obtained from Charles River (Raleigh, N.C.),were used for assessment in hot plate nociception, rectal temperature,and ring immobility assays. Separate mice were used for testing eachdose of each compound in this battery of procedures. The mice had freeaccess to food and water when in their home cages. All animals were keptin a temperature-controlled (20-22° C.) environment with a 12-hourlight-dark cycle (lights on at 7 a.m.).

In Vivo Methods

Each mouse was tested in a battery of three tests, in which cannabinoidCB1 agonists produce in vivo effects in mice (Martin et al., 1991):antinociception (hot plate assay), decreased rectal temperature and ringimmobility. Prior to administration of the test compound, rectaltemperature and baseline latency in the hot plate test were measured inthe mice. The latter procedure involved placing the mouse on a heatedsurface (mouse cold/hot plate analgesia apparatus; Stoelting, Wood Dale,Ill.) at a setting of 55° C. Time until the mouse lifts or licks a pawwas measured, whereupon the mouse was removed from the apparatus. If themouse did not lift or lick a paw within 30 sec, it was removed from theapparatus and a latency of 30 sec was recorded. After measurement ofbaseline temperature and hot plate latency, mice were administeredvehicle or drug via oral gavage. Hot plate latency and temperature weremeasured again at 90 minutes after administration (via oral gavage) ofpeanut oil vehicle or JBT-101 or at 60 minutes after administration (viaoral gavage) of Δ9-THC or indomethacin. Subsequently, the mice wereplaced on a 5.5 cm ring attached at a height of 16 cm to a ring stand,and the amount of time the animals remained motionless during a 5 minperiod was recorded. In addition, the number of times the mouse fell orjumped off the ring was recorded. If the mouse fell off the ring morethan 5 times, the test was terminated.

Data Analysis.

Rectal temperature values were expressed as the difference betweencontrol temperature (before injection) and temperature following drugadministration (Δ° C.). Antinociception was expressed as the percentmaximum possible effect (MPE) using a 30-s maximum test latency asfollows: [(test−control)/(30−control)]×100. During assessment forcatalepsy, the total amount of time (in seconds) that the mouse remainedmotionless on the ring apparatus (except for breathing and whiskermovement) was measured and was used as an indication of catalepsy-likebehavior. This value was divided by 300 s and multiplied by 100 toobtain a percent immobility. If the mouse fell off the ring more than 5times, the test was terminated and ring immobility data for the mousewere not included in analysis. Separate between-subjects ANOVAs wereused to analyze each measure. Significant differences from control(vehicle) were further analyzed with Tukey post hoc tests (α=0.05) asnecessary.

In Vivo Tests: Results and Discussion

Control tests revealed that peanut oil vehicle (negative control) wasnot active in any of the three tests (hot plate, rectal temperature andcatalepsy) [FIGS. 14, 15 and 16, left side of each panel]. A 30 mg/kgdose of indomethacin also did not significantly affect any of the threemeasures. In contrast, at oral doses of 30 and/or 100 mg/kg, Δ9-THC(positive control) produced significant antinociception [100 mg/kg only;F(9,50)=5.71, p<0.05. FIG. 15], ring immobility [both doses;F(9,48)=21.18, p<0.05. FIG. 14], and hypothermia [both doses;F(9,50)=15.08, p<0.05. FIG. 16], with each measure compared to vehiclecondition. JBT-101 was assessed in the three in vivo tests at oral dosesranging from 0.05-56 mg/kg. None of the doses tested producedsignificant antinociception in the hot plate test (FIG. 15, right side)or change in rectal temperature (FIG. 16, right side). At oral doses of30 and 56 mg/kg, JBT-101 significantly increased percent ring immobilityin the catalepsy test, as compared to vehicle condition (FIG. 14, rightside. F(9,48)=21.18, p<0.05]. The magnitude of increase was similar at30 and 56 mg/kg doses and approximated the increase observed at the 30mg/kg dose of Δ9-THC. Lower doses of JBT-101 (up to 10 mg/kg) did notaffect this measure.

In summary, the pattern of effects produced by JBT-101 (0.05-56 mg/kg)in the hot plate and rectal temperature tests in mice did not resemblethose exhibited by Δ9-THC (present study; Martin et al., 1991) and otherpsychoactive cannabinoids, including aminoalkylindoles (Compton et al.,1992a), bicyclic cannabinoids (Compton et al., 1992b), and indole- andpyrrole-derived cannabinoids (Wiley et al., 1998; Wiley et al., 2012).Although JBT-101 increased ring immobility at higher doses (30 and 56mg/kg), with a magnitude similar to that produced by 30 mg/kg Δ9-THC,its overall pattern of pharmacological effects did not suggestΔ9-THC-like psychoactivity.

Summary

The profile of pharmacological effects produced by JBT-101 (ultrapureajulemic acid) differs significantly from previously reported effects ofajulemic acid. Previously synthesized (non-purified) ajulemic acid hasshown efficacy in several preclinical models of pain and inflammation(reviewed in Wiley, 2005); however, it also produced a profile ofpharmacological effects in vivo that is characteristic of Δ9-THC andother psychoactive CB1 receptor agonists. These effects includedsuppression of spontaneous activity, antinociception, hypothermia, andcatalepsy in mice and Δ9-THC-like discriminative stimulus effects inrats (Vann et al., 2007). These effects are consistent with the good CB1receptor binding affinities exhibited by these earlier ajulemic acidsynthesis products: K_(i)=5.7 nM for Novartis compound (Dyson et al.,2005) and K_(i)=32.3 nM for HU-239 (Pertwee et al., 2010). Further, theratio of CB1/CB2 binding for these compounds was low (0.10 and 0.19,respectively). In contrast, JBT-101 showed more than 12-fold selectivebinding affinity at the CB2 receptor (K_(i)=51±11 nM) as compared to theCB1 receptor (K_(i)=628±6 nM). As shown herein, JBT-101 also exhibitedsimilar selectivity for activation of CB2 receptors as compared to CB1receptors. Furthermore, at doses up to 56 mg/kg (p.o.), behavior effectsthat are characteristically observed following administration of Δ9-THCand other psychoactive cannabinoids were minimally observed withultrapure ajulemic acid. While JBT-101 increased ring immobility, it didso only at higher doses (30 and 56 mg/kg). Together, these resultsdemonstrate that the effects of JBT-101 differ from those of earliersyntheses of (non-purified) ajulemic acid. In conclusion, thepharmacological profile of JBT-101 is consistent with it being a CB2selective compound with little CB1 receptor activity.

Example 4 Binding Curves of Selected Cannabinoids for CB2 and CB1

FIG. 12 shows the binding curves for ultrapure AJA and referencecannabinoid antagonists selective for CB2 and CB1.

CB Receptor Binding Assays

Membrane preparation-HEK-293T cells were cultured according to ATCC(Manassas, Va.) guidelines and transfected with human CB1 cDNA (GenbankX54937) or CB2 cDNA (Genbank X74328), operably linked to the SV40promoter, using Polyfect (Qiagen, Valencia, Calif.) or Fugene (Roche,Nutley, N.J.) according to manufacturer's instructions. 48 h aftertransfection, cells were harvested in ice cold membrane buffer (20 mMHEPES, 6 mM MgCl2, 1 mM EDTA, pH 7.2) using a cell scraper. Cells weretransferred to a nitrogen cavitation chamber and a pressure of 900 barwas applied for 30 min. The pressure was released and the cell debriswas collected and centrifuged at 1000 g at 4° C. for 10 min. Thesupernatant was collected and the spin was repeated until thesupernatant was free of precipitate. Membranes were then pelleted bycentrifugation at 12,000 g at 4° C. for 20 min. Membranes wereresuspended in an appropriate amount of membrane buffer. The membraneconcentration was determined using a BioRad (Hercules, Calif.) proteinassay dye reagent according to manufacturer's instructions. Membraneswere diluted to 1 mg/ml and aliquots snap-frozen in liquid nitrogen andstore at −80° C.

Binding assay—0.5-10 ng of membranes expressing human CB1 or human CB2receptors were incubated in binding buffer (50 mM Tris, 10 mM MgCl₂, 1mM EDTA, 0.1% BSA, pH 7.4) in the presence of 0.5 to 2 nM radioligand([3H]-CP55940; Perkin Elmer except 3 nM [3H]-SR141716 was used as theradioligand where noted), and varying concentrations of ligands (totalvolume 200 μL/well of a 96 well plate). Membranes were incubated for 2hr at room temperature, then filtered onto a presoaked (with 0.1%polyethylenimine for 1 to 2 hr) 96 well GF/B filterplate (PackardBioscience, Shelton, Conn.) and washed with 500 mL ice cold wash buffer(25 mM HEPES, 1 mM CaCl₂), 5 mM MgCl₂, 0.25M NaCl) using a Filtermate196 Harvester (Packard Instruments, Downers Grove, Ill.). The filterplates were dried before addition of 50 μL of scintillation fluid toeach well (Microscint 20, Packard, Shelton, Conn.). Plates were countedon a Topcount NXT (Packard, Shelton, Conn.).

Data Analysis—Graphs were plotted and IC₅₀ values were determined bynonlinear regression analysis using Prism software (GraphPad version4.0, San Diego, Calif., USA). Ki values were calculated from IC₅₀ valuesusing the Cheng & Prussoff method using reported Ki values of 2.9 nM forSR141716 receptors for human CB1 receptors, and 2.5 nM and 0.92 nM forCP55,940 for human CB1 and human CB2 receptors, respectively (McPartlandet al, BJP, 2007).

Example 5 Ultrapure AJA Induced CB2 Receptor-Mediated In Vivo EffectsFibrosis Animal Model—Bleomycin Induced Skin Fibrosis

Groups of 8 mice (6-12 week-old C57B1) received daily s.c. injections ofbleomycin (20 ug/mouse) or vehicle for 14 days, followed by one weeks ofrecovery. In parallel, starting day 1, ultrapure AJA suspended in 2%methylcellulose (MC) or vehicle was administered by oral gavage at 0,2.5, 5.0 and 10 mg/kg. On day 21, mice were sacrificed. Their skin washarvested and processed for routine histology (H&E, trichrome andpicrosirius red stains), immunohistology (paraffin-embedded or frozensamples), in situ hybridization, collagen (SIRCOL) assays, and RNAisolation for real-time qPCR or microarray hybridization. Skin sectionswere carefully characterized and quantified for dermal inflammation,dermal thickening, collagen accumulation (Trichrome), and collagencross-linking (Sirius red) in lesional skin.

Results: As seen in FIG. 18, ultrapure AJA was effective at all dosestested for inhibition of CB2 mediated skin fibrosis in the mousebelomycin model. These same doses were completely ineffective in the CB1mediated behavioral models shown in FIGS. 14-16 supporting themaintenance of CB2 activity in ultrapure ajulemic acid in the absence ofany CB1 activity.

Inflammation Animal Model—Paw Edema Model

CD-1 Mice (Female) were randomly allocated to experimental groups andallowed to acclimatise for one week. Prior to test compoundadministration, baseline right hind paw volume was measured under gas(isoflurane) anaesthesia using a water displacement device(plethysmometer, Stoelting). On Day 0, ultrapure AJA suspended in 2% MCor vehicle was administered at 0, 5, 50 and 500 ug/kg by oral gavage.Ninety minutes after treatment administration, the animals were given 10microliters of a 100 mg/ml arachidonic acid solution in 5% ethanol bysubcutaneous injection into the plantar aspect of the right hind paw. Acontrol group of animals (Group 1) was administered with an equivalentvolume of a 5% ethanol solution. The left hind paws were not injected.Intra-plantar injections were performed under gas anaesthesia. 45minutes after the intra-plantar injections right hind paw volume wasmeasured under gas anaesthesia using a plethysmometer.

Results: As seen in FIG. 19, ultrapure AJA was effective at all dosestested at inhibiting inflammation in the mouse paw edema model. Thesesame doses were completely ineffective in the CB1 mediated behavioralmodels shown in FIGS. 14-16 supporting the maintenance of CB2 activityin ultrapure ajulemic acid in the absence of any CB1 activity.

REFERENCES

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1. A pharmaceutical composition comprising an enterically coated tabletcontaining (i)(6aR,10aR)-3-(1′,1′-dimethylheptyl)-Δ8-tetrahydro-cannabinol-9-carboxylicacid, or a pharmaceutically acceptable salt thereof, (ii) an opiate, and(iii) a pharmaceutically acceptable excipient.
 2. A method of treatingpost-operative adhesions in a subject in need thereof, said methodcomprising topically administering to the subject a solution comprising(i)(6aR,10aR)-3-(1′,1′-dimethylheptyl)-Δ8-tetrahydro-cannabinol-9-carboxylicacid, or a pharmaceutically acceptable salt thereof, (ii) ananti-inflammatory agent, and (iii) a carrier comprising mineral oil. 3.A method of treating a human subject having a fibrotic disease, themethod comprising administering once daily, twice daily, or three timesdaily to the subject an effective amount of a pharmaceutical compositioncomprising a preparation of ajulemic acid and a pharmaceuticallyacceptable excipient, wherein the preparation of ajulemic acid comprisesat least 98% (w/w) ajulemic acid or a pharmaceutically acceptable saltthereof, wherein the preparation of ajulemic acid has a Ki for the CB1receptor (Ki CB1) ranging from 10 times to 40 times greater than its Kifor the CB2 receptor (Ki CB2), and wherein the Ki CB1 and the Ki CB2 areeach determined according to a competitive radioligand binding assaywith [³H]-CP55940 as the radioligand.
 4. The method of claim 3, whereinthe fibrotic disease is selected from the group consisting ofscleroderma, systemic sclerosis, sine scleroderma, a scleroderma-likedisorder, liver cirrhosis, interstitial pulmonary fibrosis, idiopathicpulmonary fibrosis, Dupuytren's contracture, keloids, cystic fibrosis,chronic kidney disease, chronic graft rejection, scarring or wouldhealing abnormalities, post-operative adhesions, reactive fibrosis,dermal fibrosis, lung fibrosis, liver fibrosis, kidney fibrosis, andheart fibrosis.
 5. The method of claim 4, wherein the fibrotic diseaseis selected from the group consisting of dermal fibrosis, scleroderma,cystic fibrosis, and idiopathic pulmonary fibrosis.
 6. The method ofclaim 3, wherein the preparation of ajulemic acid has Ki CB1 rangingfrom 10 times to 20 times greater than its Ki CB2.
 7. The method ofclaim 3, wherein the preparation of ajulemic acid comprises at least 99%(w/w) ajulemic acid.
 8. The method of claim 3, wherein the preparationof ajulemic acid comprises less than 0.5% (w/w) of11-hydroxy-(6aR,10aR)-3-(1′,1′-dimethylheptyl)-Δ8-tetrahydrocannabinol(HU-210).
 9. The method of claim 8, wherein the preparation of ajulemicacid comprises less than 0.1% (w/w) of11-hydroxy-(6aR,10aR)-3-(1′,1′-dimethylheptyl)-Δ8-tetrahydrocannabinol(HU-210).
 10. The method of claim 3, wherein the pharmaceuticalcomposition is administered orally, intravenously, topically,interstitially, by inhalation, via an implant, via a patch, or byophthalmic administration.
 11. The method of claim 3, wherein thepharmaceutical composition is in the form of a capsule.
 12. The methodof claim 11, wherein the tablet or the capsule comprises from 5 mg to180 mg of the preparation of ajulemic acid or a pharmaceuticallyacceptable salt thereof.
 13. A method of treating a human subject havingan inflammatory disease, the method comprising administering once daily,twice daily, or three times daily to the subject an effective amount ofa pharmaceutical composition comprising a preparation of ajulemic acidand a pharmaceutically acceptable excipient, wherein the preparation ofajulemic acid comprises at least 98% (w/w) ajulemic acid or apharmaceutically acceptable salt thereof, wherein the preparation ofajulemic acid has a Ki for the CB1 receptor (Ki CB1) ranging from 10times to 40 times greater than its Ki for the CB2 receptor (Ki CB2), andwherein the Ki CB1 and the Ki CB2 are each determined according to acompetitive radioligand binding assay with [³H]-CP55940 as theradioligand.
 14. The method of claim 13, wherein the inflammatorydisease is selected from the group consisting of systemic lupuserythematosus, AIDs, multiple sclerosis, rheumatoid arthritis,psoriasis, diabetes, cancer, asthma, atopic, dermatitis, an autoimmunethyroid disorder, ulcerative colitis, Crohn's disease, stroke, ischemia,and a neurodegenerative disease.
 15. The method of claim 14, wherein theinflammatory disease is systemic lupus erythematosus.
 16. The method ofclaim 13, wherein the preparation of ajulemic acid has Ki CB1 rangingfrom 10 times to 20 times greater than its Ki CB2.
 17. The method ofclaim 13, wherein the preparation of ajulemic acid comprises at least99% (w/w) ajulemic acid.
 18. The method of claim 13, wherein thepreparation of ajulemic acid comprises less than 0.5% (w/w) of11-hydroxy-(6aR,10aR)-3-(1′,1′-dimethylheptyl)-Δ8-tetrahydrocannabinol(HU-210).
 19. The method of claim 18, wherein the preparation ofajulemic acid comprises less than 0.1% (w/w) of11-hydroxy-(6aR,10aR)-3-(1′,1′-dimethylheptyl)-Δ8-tetrahydrocannabinol(HU-210).
 20. The method of claim 13, wherein the pharmaceuticalcomposition is administered orally, intravenously, topically,interstitially, by inhalation, via an implant, via a patch, or byophthalmic administration.
 21. The method of claim 13, wherein thepharmaceutical composition is in the form of a capsule.
 22. The methodof claim 21, wherein the tablet or the capsule comprises from 5 mg to180 mg of the preparation of ajulemic acid or a pharmaceuticallyacceptable salt thereof.